Fig 1.
Experimental assessment of infectious phenotype of spirochetes in fed nymphs.
Ixodes scapularis nymphs (infected as larvae with B. burgdorferi strain B31) were fed on naïve mice, on mice infected with the homologous strain (B31), or on mice infected with a heterologous strain (PKo) and collected after feeding to repletion. Blood-meal hosts representing different strains of wild-type and immune-deficient laboratory mice were used in separate experiments. A.) OspC+ spirochetes in the midguts of a subset of fed ticks from each mouse/tick cohort were visualized by immunofluorescence assay (IFA) with a monoclonal antibody specific for OspC and a polyclonal anti-B. burgdorferi serum to counter-stain midgut spirochetes B.) Viable spirochetes in homogenates prepared from the remaining fed ticks (pooled) in each mouse/tick cohort were quantified by plating an aliquot for colony forming units (CFU). C.) Mice were injected with serial dilutions containing defined numbers of viable spirochetes from fed tick homogenates to assess the relative infectivity of each experimental group.
Fig 2.
Spirochete burden in infected nymphs fed on naïve and infected wild-type mice.
Cohorts of nymphs infected as larvae with B. burgdorferi strain B31 were fed to repletion on groups of naïve, heterologously-infected (PKo) or homologously-infected (B31) mice, as identified at the bottom of the graph. Fed nymphs were collected at drop-off, pooled for individual mouse/tick cohorts, and crushed. The number of viable spirochetes per tick was estimated by plating an aliquot of each pooled homogenate for colony forming units (CFU). Each point on the graph represents the average spirochete load per tick for each cohort of 5–12 infected nymphs fed upon individual mice, with a total of 6–8 animals per group. (**)P = 0.02 naïve versus homologous; (*)P = 0.1 heterologous versus homologous; P = 0.8 naïve versus heterologous; P values calculated using non-parametric rank order test.
Table 1.
Infectivity of spirochetes in infected nymphs after feeding on naïve or infected WT mice.
Fig 3.
OspC production by spirochetes in infected nymphs fed on naïve or infected wild-type mice.
Dissected midguts of strain B31-infected nymphs fed on naïve, heterologously infected (strain PKo) or homologously infected (strain B31) mice, as identified to the right of the images, were co-stained with a rabbit polyclonal anti-B. burgdorferi serum and a mouse monoclonal antibody that recognizes OspC. Primary antibody binding, as identified above the panels, was visualized on a fluorescent microscope (20X magnification) with TRITC- (total B. burgdorferi) and FITC- (OspC+ B. burgdorferi) tagged secondary antibodies. The entire experiment and IFA analysis were conducted 4 times, with visual assessment of 6 nymphs per group, and 5 fields per nymph. Representative IFA images are shown.
Fig 4.
Spirochete burden in infected nymphs fed on naïve and infected Rag1 KO mice.
Cohorts of nymphs infected as larvae with B. burgdorferi strain B31 were fed to repletion on groups of naïve, heterologously infected (PKo) or homologously infected (B31) Rag1 KO mice, as identified at the bottom of the graph. Fed nymphs were collected at drop-off, pooled for individual mouse/tick cohorts, and crushed. The number of viable spirochetes per tick was estimated by plating an aliquot of each pooled homogenate for colony forming units (CFU). Each point on the graph represents the average spirochete load per tick for each cohort of 5–12 infected nymphs fed upon individual mice, with a total of 2 animals per group. The number of viable spirochetes per tick was estimated by plating an aliquot of the pooled homogenate of crushed ticks from each mouse/tick cohort for colony forming units (CFU). Naïve versus homologous P = 0.3; heterologous versus homologous P = 0.3; naïve versus heterologous P = 0.6; calculated using non-parametric rank order test.
Table 2.
Infectivity of spirochetes in infected nymphs fed on naïve and infected Rag1 KO mice.
Table 3.
Infectivity of spirochetes in infected nymphs fed on naïve and infected muMT- mice.
Table 4.
Infectivity of spirochetes in tick homogenates after exposure to immune serum.
Fig 5.
Serologic response of infected mice to homologous and heterologous B. burgdorferi strains.
A.) Representative immunoblots with sera of mice infected with B. afzelii strain PKo (lanes 1–5) or B. burgdorferi strain B31 (lanes 6–10) against whole cell lysates of strains PKo (top panels) and B31 (bottom panels) B.) Whole cell lysates of strains B31 (lanes 1–2) and PKo (lanes 3–4), with or without OspC (lanes 1 & 3 versus 2 & 4, respectively), stained with coomassie brilliant blue (CBB) to visualize all proteins (left-most panel), or transferred to membranes and incubated with infected mouse sera (panels on the right side of the figure). A blot containing the same lysates was also incubated with polyclonal rabbit antiserum raised against recombinant OspC from strain B31 (second panel from the left). The mobility of OspC is indicated at the right side of the figure. Molecular weight markers (MW) are visible at the left side of the figures, with mass indicated (kD). C.) ELISA titers of pooled sera from 5 mice infected with either strain B31 or PKo and tested against lysates of homologous and heterologous strains, as identified beneath the graphs. Each bar represents the average of three technical replicates, with the standard deviation shown. Baseline absorbances were determined for the same dilutions of pooled pre-immune sera, in triplicate, against B31-S9 and PKo lysates. The threshold for positive sero-reactivity was set at 3 standard deviations above the mean absorbance of pre-immune sera at each dilution, indicated by a dark line. The ELISA titer represents the highest dilution of immune sera at which absorbance above this baseline cut-off was achieved, as indicated by asterisks.
Fig 6.
Recognition of heterologous spirochetes in infected ticks by immune mouse sera.
Dissected midguts of strain B31-infected nymphs fed on naïve mice were co-stained with a rabbit polyclonal anti-B. burgdorferi serum and sera of mice infected with strain PKO (heterologous infection, middle row) or strain B31 (homologous infection, bottom row); uninfected mouse sera was used as a negative control (top row). Primary antibody binding, as identified above the panels, was visualized on a fluorescent microscope (20X magnification) with TRITC- (anti-rabbit Ig) and FITC- (anti-mouse Ig) tagged secondary antibodies, as shown in left and middle columns, respectively. Merged TRITC- and FITC images are shown in the right column.
Fig 7.
Schematic representation of the impact of host immunity on the infectivity of Lyme disease spirochetes within feeding ticks.
Tick-borne spirochetes become highly infectious when the vector feeds upon an uninfected host (naïve mouse), or upon infected hosts lacking neutralizing antibodies (heterologously infected or immune-deficient). In sharp contrast, tick-borne spirochetes are non-infectious when the vector feeds upon an immune-competent host infected with the same B. burgdorferi strain (homologously infected and immune-competent). This outcome indicates that strain-specific antibodies neutralize infectious organisms within the tick midgut, prior to transmission, when the mammalian host and tick vector are infected with the same Lyme disease spirochete strain.