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* Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, NY 12201; and
Wadsworth Center, New York State Department of Health, Albany, NY 12208
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One approach to identifying the relevant mechanism of humoral immunity is to identify characteristics of Abs that are critical for efficacy in vivo. A previous study identified and characterized effective IgG2a and IgG3 outer membrane protein (OMP)3-specific Abs (5), but it was not clear to what extent properties such as epitope, isotype, and affinity contributed to Ab efficacy. The present study extends previous work by evaluating the in vivo efficacy of a large panel of OMP-specific mAbs. The findings reveal important characteristics of Abs that are most likely critical for effectiveness during this intracellular infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 and BALB/c-scid mice were obtained from The Jackson Laboratory (Bar Harbor, ME), or were bred in the Wadsworth Center Animal Care Facility, under institutional guidelines for animal care and use. Six- to 12-wk-old, sex-matched mice were routinely infected via the peritoneum with 1–2 x 106 E. chaffeensis-infected DH82 cells, as described previously (6). Institutional animal care and use guidelines do not permit the use of death as an experimental endpoint in these studies, so body weight measurements were used to monitor animal health and Ab efficacy (see below). The infected animals were sacrificed when moribund, as characterized by a lack of mobility, hunched posture, ruffled fur, and a pronounced loss of body weight (>30% loss of initial weight). Tissue samples were harvested and stored at -80°C before further analysis.
Hybridoma production and Ab purification
Three fusions for hybridoma production were performed independently. C57BL/6 mice were infected via the peritoneum with E. chaffeensis-infected DH82 cells, in the absence of adjuvant. For the first and second fusions, mice were infected two to four times at 2- to 4-wk intervals. For the third fusion, mice were infected once, and the fusion was performed 2 wk later. Splenocytes were harvested from the infected mice and fused to the myeloma cell line SP2/0, using standard protocols. The hybridoma supernatants were screened for reactivity to E. chaffeensis by immunofluorescence assay, as described previously (2), and hybridomas that produced specific Abs were expanded and subcloned by limiting dilution. H and L chain Ab isotypes were determined by ELISA using isotype-specific polyclonal reagents (Southern Biotechnology Associates, Birmingham, AL). Abs were purified from hybridoma culture supernatants by fast performance liquid chromatography, using protein A- or G-Sepharose (for IgGs; Amersham Pharmacia Biotech, Piscataway, NJ) or IgM purification columns (for IgM; Amersham Pharmacia Biotech), following the instructions of the manufacturer. The concentrations of purified Abs were determined by measurement of absorbance at 280 nm using a spectrophotometer (Pharmacia Biotech, Cambridge, U.K.). Unpublished data indicated that a single dose of 50 µg mAb Ec56.5, administered on day 10 postinfection, was sufficient to mediate nearly complete bacterial clearance within 4 days. Abs were administered via the peritoneum in weekly doses of 200 µg to facilitate protection in the long-term studies.
ELISA
Epitope analyses were performed as described previously (5). Purified rOMP-1g and truncated rOMPs were adsorbed overnight to 96-well microtiter plates (Dynex Technologies, Chanitilly, VA) at a concentration of 3 µg/ml in PBS, and peptides were adsorbed overnight in sodium carbonate buffer (pH 9.6) at a concentration of 10 µg/ml. The microtiter plates were blocked with 1% nonfat dry milk in PBS. Bound Abs were detected using alkaline phosphatase-conjugated anti-mouse Ig secondary Abs (Southern Biotechnology Associates), followed by p-nitrophenyl phosphate (Sigma-Aldrich, St. Louis, MO). The absorbance was read at 405 nm with a ThermoMax microplate reader (Molecular Devices, Sunnyvale, CA).
Ab serum t1/2 measurements
For determination of Ab t1/2 in serum, uninfected BALB/c-scid mice were administered a single dose of 200 µg Ab via the peritoneum, and 100 µl blood samples were withdrawn retroorbitally 1, 4, and 7 days after Ab administration. Sera were obtained after centrifugation of blood samples at 4000 rpm for 20 min. The concentrations of mAb in the sera were determined by ELISA, using predetermined concentrations of purified Abs as standards. Abs used as standards for quantitation were identical with those used for in vivo administration.
Affinity measurements
Affinity measurements were performed with the BIAcore 3000
instrument (BIAcore, Uppsala, Sweden). Purified rOMP-1g and purified
rabbit anti-mouse Fc
were covalently bound to the flow cell
surfaces of CM5 sensor chips (BIAcore), using an amine coupling kit
supplied by the manufacturer. HEPES-buffered saline (10 mM HEPES, 150
mM NaCl, 3.4 mM EDTA, and 0.05% surfactant P20, pH 7.4) was used as
the standard running buffer. Serial dilutions of mAb (1000, 500, 250,
125, 62.5 nM) were injected over four flow cell surfaces containing
either rOMP-1g, anti-Fc, or a blank control treated with
coupling reagents, or an unmodified blank control. The injection volume
was 30 µl, with flow rate of 15 µl/min. Ab dissociation in running
buffer was monitored for 4.7 min, and the flow cells were regenerated
after each assay with 100 mM HCl/50 mM glycine, pH 2.4. The responses
were measured in resonance units (RU). Association and dissociation
rates were calculated from RU data using the BIAevaluation software
supplied by the manufacturer.
Quantitative PCR analyses
The Ehrlichiae were quantitated by quantitative real-time PCR using the SYBR Green PCR Reagent kit (PE Applied Biosystems, Foster City, CA), and the following E. chaffeensis-specific 16S rDNA oligonucleotide primers: 5'-AACACATGCAAGTCGAACGG-3' (sense) and 5'-CCCCCGCAGGGATTATACA-3' (antisense) at a concentration of 100 nM. Genomic DNA (5 ng) was amplified for 40 cycles (95°C/15 s; 60°C/60 s) using 0.025 U/µl AmpliTaq Gold DNA polymerase (PE Applied Biosystems), in reaction buffer containing 3 mM MgCl2 and 200 nM dNTPs, in a volume of 25 µl. Reaction products were monitored in real time using the ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). A standard curve was generated using data from analysis of standardized quantities of a gel-purified DNA fragment that contained nt 1–619 of the 16S-rDNA gene of E. chaffeensis. Quantitation of the template standards was performed using a Picogreen DNA quantitation kit (Molecular Probes, Eugene, OR). The data were analyzed using the software supplied by the manufacturer (PE Applied Biosystems).
Evaluation of Ab efficacy in vivo
Ab efficacy was evaluated in long-term studies by measurement of body weight changes. Purified Abs (200 µg) were injected via the peritoneum into BALB/c-scid mice on day 7 postinfection, and at weekly intervals thereafter. The mice were monitored for disease and morbidity throughout the experiments, and the changes in body weight from day 0 were recorded. In each experiment, buffer was administered as a negative control, and the protective Ab Ec56.5 was used as a positive control. In some experiments, uninfected SCID mice were also included in the analyses.
To quantitate Ab efficacy data, and to compare data from multiple experiments, the value of mean integrated weight difference (MIWD) was determined for each Ab-treated group to indicate the in vivo efficacy of each Ab. The body weight changes in groups of Ab-treated vs untreated infected mice were first normalized by integrating the body weight data, over time, up to the point that the untreated mice were deemed moribund. The difference in integrated values between the Ab-treated and untreated control groups was then divided by the number of days to morbidity, to obtain the MIWD values (in grams) for each group of Ab-treated mice. The weight data integration was performed using KaleidaGraph data analysis/graphing application software (Synergy Software, Reading, PA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For production of mAbs for study of the humoral immune response,
immunocompetent C57BL/6 mice were infected via the peritoneum with
E. chaffeensis (the Arkansas isolate), spleen cells were
isolated at various times thereafter, hybridomas were generated, and
Abs were screened by immunofluorescence assay. A total of 100 E.
chaffeensis-specific hybridomas was recovered and characterized.
The hybridomas described in this study were recovered from three
separate hybridoma fusions. Two fusions were performed following
repeated infections (fusions 1 and 2), and a third 14 days after a
single infection (fusion 3; Table I
). Two
Abs from the panel have been described previously, Ec56.5 (IgG2a) and
Ec18.1 (IgG3). Both Abs were effective in infected SCID mice, mediating
significant clearance of the infection from liver tissues within 4 days
of administration (5).
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). IgM and all subclasses of IgG were recovered,
except IgG1, even though OMP-specific IgG1 polyclonal serum responses
were identified in C57BL/6 mice (data not shown). The IgGs were
predominant among those Abs that were OMP specific (75%), and IgM were
predominant among the non-OMP-specific Abs (57%; data not shown). Highly restricted epitope distribution of OMP-1g Abs
The predominance of OMP Abs in the humoral response, as indicated by the hybridoma analyses, was consistent with that observed in studies of the polyclonal Ab response, which suggested that the specificity of the Ab panel was indeed representative of the overall humoral response during infection of C57BL/6 mice. Further characterization of the OMP-specific Abs was performed, because OMP-specific Abs were shown previously to provide protection from lethal infection in SCID mice (5).
The E. chaffeensis OMPs together form a family of 21 related
proteins that differ largely in three short regions, called
hypervariable regions (HVRs) (8, 9). To identify the
epitopes of the panel of OMP-specific Abs, a series of truncated
rOMP-1g Ags, each lacking one or more of the three HVRs (OMP
3,
2/3, 1/2/3, and 1), were analyzed by ELISA, as described
previously (5). All but one of the OMP-specific Abs (39 of
40) recognized rOMP-1g Ags that lacked HVRs 3 and 2 (OMP3, 2/3),
but most failed to recognize those lacking HVR1 (OMP1/2/3 or 1;
Table II). These observations indicated
that most Abs recognized an epitope within the first HVR. Moreover,
most Abs also failed to recognize a related OMP, OMP-1d (Table II),
indicating that these Abs most likely recognized residues in HVR1 that
differed between OMP-1g and OMP-1d. This pattern of epitope specificity
was similar to that described previously for the Abs Ec56.5 and Ec18.1,
so additional analyses of the remaining OMP Abs were performed using
synthetic peptides derived from HVR1. Seventy-two percent (29 of 40) of
the OMP-specific Abs recognized the OMP-1g peptide 61–90, and 32% (13
of 40) recognized nested peptide containing residues 65–78 (Table II).
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Evaluation of Ab efficacy
To identify Abs that could mediate host defense, 16 of the 40
OMP-specific Abs were chosen for in vivo study, including the two
previously described Abs, Ec56.5 and Ec18.1 (5). Abs were
chosen for in vivo analyses on the basis of their class (IgM and IgG),
subclass (IgG2a, IgG2b, and IgG3), and epitope specificity (Table III). Abs of common isotype were
grouped operationally, based on their ability or inability to recognize
HVR1 peptide 61–90. Purified Abs (200 µg) were injected via the
peritoneum into susceptible immunodeficient BALB/c-scid mice
after infection had been established (7 days postinfection), and at
weekly intervals thereafter. The mice were monitored for disease and
morbidity throughout the experiments. Effective Abs have been shown
previously to mediate partial to apparently complete bacterial
clearance from liver tissue within as early as 4 days post-Ab
administration, whereas isotype-matched irrelevant control Abs or PBS
had no effect (5). Fully quantitative assays had been
unavailable, however, so to refine the quantitation of Ab
efficacy in the long-term treated BALB/c-scid mice used in
this study, a quantitative real-time PCR assay for E.
chaffeensis 16S rDNA was developed and used (for details, see
Materials and Methods). Weekly treatment of mice with Ec56.5
led to a 3500-fold reduction in bacterial colonization in the liver
(Fig. 1), supporting our previous study.
Bacterial infection was also decreased 100- to 400-fold in the spleen,
peritoneal exudate, and peripheral blood (Fig. 1). These data indicate
that the Abs had profound systemic effects on bacterial colonization in
the animals that had undergone periodic Ab treatment.
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. In each experiment, buffer was
administered as a negative control because previous studies
demonstrated that irrelevant isotype-matched Abs were ineffective.
Ec56.5, which was previously shown to be effective, was used as a
positive control. The data in Fig. 2 are expressed as the relative
change in body weight from day 0, to control for differences in
starting body weight among the mice. In some experiments, uninfected
SCID mice were also included in the analyses.
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, a and
b). Other Abs were ineffective (e.g., Ec12.7, Ec13.1; Fig. 2, a and d), or only partially effective (e.g.,
Ec18.1, Ec104.2; Fig. 2, a and c). Mice treated
with the highly effective Abs remained healthy. Partially effective Abs
delayed, but did not prevent, the onset of morbidity (Fig. 2, Table III).
To quantitate Ab efficacy data, and to compare data from multiple
experiments, body weight changes in groups of Ab-treated vs untreated
infected mice were normalized. This was performed by integrating the
body weight data, over time, up to the point that the untreated mice
were deemed moribund. The difference in integrated values between
the Ab-treated and untreated control groups was then divided by the
number of days to morbidity, to obtain the MIWD (in grams) for each
group of Ab-treated mice (Table III). Highly effective Abs, initially
identified by their ability to prevent morbidity in SCID mice,
exhibited MIWD values greater than 1.5 g. Partially effective Abs,
which delayed morbidity and prolonged survival, exhibited MIWD values
between 0.5 and 1.5 g. Abs that exhibited MIWD values less than
0.5 g were considered ineffective.
The data also revealed that among IgG Abs of identical subclass, higher
efficacy was correlated with recognition of the OMP HVR1 peptide
61–90. Within this group, the IgG2a were highly effective, followed by
the partially effective IgG3 and IgG2b. None of the IgM was effective.
Differences in Ab efficacy among Abs of different isotypes were not due
to significant differences in serum
t1/2, as measurements revealed similar
t1/2 of 1.2–3 days for IgG2a, IgG2b,
and IgG3 in SCID mice (Table III). The serum
t1/2 of the Abs measured in this study
in SCID mice were much shorter than those measured in normal mice
(4–8 days for IgG2a, IgG2b, and IgG3) (10). It is known
that the rate of IgG metabolism is inversely correlated to the serum
concentration of IgG (11), so the shorter serum
t1/2 of IgG subclasses in SCID mice
may be due to faster catabolism of Abs in mice lacking endogenous Ig.
Despite the shorter t1/2 in vivo,
serum Ab concentrations in SCID mice ranged, for Ec56.5 (IgG2a), from
33 µg/ml (212 nM) on day 1 to 9 µg/ml (57.7 nM) on day 7 post-Ab
administration, so reasonably high concentrations of the administered
Abs were most likely available during the weekly interval between Ab
administrations (data not shown).
Effective Abs were of extraordinarily high affinity
Although the above data suggested that both isotype and epitope
were important correlates for Ab efficacy, it was also possible that
affinity differences were critical. To evaluate the contribution of Ab
affinity, equilibrium-binding constants and binding
t1/2 were determined by surface
plasmon resonance, using highly purified rOMP-1g (Fig. 3). The results revealed striking
affinity differences among the Ab panel. The most highly effective Abs,
all IgG2a, exhibited picomolar affinity constants and binding
t1/2 of 3–28 days (Table III). In
contrast, Abs of lower or no efficacy exhibited affinities in the
micromolar to nanomolar range, and binding
t1/2 that ranged from minutes to
hours. Therefore, high affinity was a critical characteristic of the
highly effective Abs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study demonstrates that mice make dominant Ab responses to E. chaffeensis OMPs, as suggested from previous studies of the polyclonal Ab responses in both mice and humans (2, 7). Forty percent of the Abs recovered from three independent hybridoma fusions recognized OMPs. Abs that recognized proteins other than OMPs were also recovered, but for the most part were low affinity IgM of undetermined specificity, and in preliminary experiments were not effective in vivo. Therefore, OMP recognition plays a major role in Ab-mediated host defense during this intracellular bacterial infection.
Fine specificity analyses indicated that nearly all of the OMP Abs studied recognized an identical or closely related epitope at the amino terminus of OMP-1g HVR1. Ab recognition of the HVR1, a region that is highly polymorphic among E. chaffeensis OMPs, supports the notion that OMP genetic diversity and antigenic recognition are correlated (12). Our previous study demonstrated that the highly effective Ab Ec56.5 required for recognition a glutamine at position 70 (5), which is substituted by a lysine residue in 10 of 21 putative OMPs of the Arkansas isolate (9). Ec56.5 did not recognize a peptide containing a glutamine to lysine substitution at position 70 (J. S. Li and G. M. Winslow, unpublished data), indicating that some of the E. chaffeensis OMPs would not be predicted to be bound by this effective Ab. Genetic variation in OMPs is therefore a potential means to generate antigenic diversity, and may allow the bacteria to evade the humoral immune response in the natural host. The data presented in this study suggest that OMP antigenic variation and subsequent immune evasion did not occur in the mouse, however, because immunity was maintained in the long-term Ab-treated animals.
Given that the OMP HVRs are highly polymorphic and that HVR1 is antigenic, it was therefore surprising that none of the OMP Abs under study in this investigation recognized HVR2, and at most one Ab recognized HVR3. This result was unlikely to be due to insufficient sampling, because 40 OMP Abs, obtained from three independent fusions, were characterized. It is also unlikely that the hybridoma-screening technique selectively identified HVR1-specific Abs. Thus, although HVR1 is highly antigenic, and is therefore likely to be exposed on the bacterial surface, polymorphic HVR2 and HVR3 may not be targets for recognition by Abs. Thus, some genetic diversity might be required for functions unrelated to immune evasion (13).
Characteristics of highly effective Abs
Three parameters were evaluated to identify properties of the Abs that were highly effective in vivo: affinity, epitope, and isotype. Although it is not known whether high affinity is essential, it was the most important correlate of efficacy. The three highly effective Abs exhibited picomolar affinities and binding t1/2 on the order of days. Abs of no or low efficacy exhibited micromolar to nanomolar affinities, and typically much shorter binding t1/2. Nevertheless, epitope recognition also appeared to influence Ab efficacy. Among Abs of identical isotype, higher efficacy was correlated with recognition of the OMP peptide 61–90. Abs that did not bind peptide 61–90 presumably recognized a conformational determinant within HVR1. It is unclear why epitope recognition may influence Ab efficacy in this model, but a possible explanation is that high affinity Abs were only generated against the linear determinant. Alternatively, fine specificity differences might modulate Ab activities mediated by other host serum components, such as complement (14).
Although the highly effective Abs were IgG2a, the requirement for isotype remains unresolved, because Abs of picomolar affinity were not recovered among other subclasses. The requirement for isotype can be best addressed using a family of IgG subclasses sharing identical V regions, but a complete family of isotype-switched variants was not recovered. However, an IgG2b isotype switch variant of a partially effective IgG3 (Ec18.1) did not exhibit increased efficacy (J. S. Li and G. M. Winslow, unpublished data), supporting the data indicating that Abs of these isotypes are equally effective. The observation that highly effective Abs were recovered as IgG2a suggests a relationship between affinity maturation and isotype switching, although additional studies would be required to resolve this possibility. The recovery of partially effective IgG3 contrasts with data from studies of Ab efficacy during intracellular infection by Cryptococcus neoformans, in which IgG3 were found to be ineffective (15). Thus, fundamentally different mechanisms of humoral immunity may be involved in the two intracellular infections.
The requirement that effective Abs exhibit high affinities might also offer one possible explanation for the apparent lack of a role for humoral immunity in many studies of host defense during other intracellular bacterial infections (16). Perhaps Abs used in adoptive transfer experiments failed to provide protection against other intracellular pathogens because Abs of appropriately high affinity were not generated, or were present in insufficient quantities. Thus, a possible involvement of Abs during other intracellular bacterial infections may require further evaluation.
Mechanisms of humoral immunity during intracellular infections
It is not yet understood how Abs mediate bacterial clearance during E. chaffeensis infection. Several studies of other intracellular pathogens have provided evidence that Abs can protect against many important intracellular bacteria, fungi, and protozoa (reviewed in Ref. 17), including Mycobacterium tuberculosis (18), Listeria monocytogenes (19, 20), Salmonella typhimurium (21, 22), Brucella abortus (23), Legionella pneumophila (24), Cryptococcus neoformans (25, 26), and Toxoplasma gondii (27). The mechanisms of humoral immunity during intracellular infection, where they are known, are highly pathogen dependent. Abs might affect the growth of some intracellular pathogens within the host cell, as has been observed during L. monocytogenes infection, in which Abs can act within infected macrophages to neutralize listeriolysin O (20). Studies of Bartonella grahamii suggest that Abs may prevent the intercellular transfer and/or subsequent invasion of intracellular bacteria (28). Alternatively, uptake of Ab-opsonized pathogens might induce an oxidative burst in phagocytes (29), or promote phagosome-lysosome fusion (30). Immune complexes of Abs and microbes or microbial products may also activate macrophages, via Fc receptors, and this may result in the elimination of intracellular pathogens, such as C. neoformans (31, 32), L. monocytogenes (33), and Leishmania major (34), through the production of reactive oxygen or nitrogen intermediates. It is not known which, if any, of these mechanisms are relevant during ehrlichial infection.
It is also not clear why picomolar affinity was an apparently critical characteristic of highly effective Abs during E. chaffeensis infection. High affinity IgG2a have been shown to be highly neutralizing during influenza infection (35), so it is possible that Abs act to opsonize Ehrlichiae released from infected cells. Perhaps the very long binding t1/2 that are correlated with high affinity are critical for efficient opsonization and/or immune complex formation. The data might also be comparable with studies of murine Ab responses against several viruses, such as influenza (36) and Ebola (37). In addition to being highly neutralizing for viral particles, IgG2a is the most efficient isotype at fixing complement (38) and for binding to Fc receptors on macrophages (39) and NK cells (40). The similar effectiveness of humoral immunity in ehrlichial and viral infections suggests that similar mechanisms may be involved.
The data presented in this study provide further support for our observations that Abs, in the absence of lymphocytes, can be highly effective during this intracellular bacterial infection. Elicitation of effective Ab responses might therefore be an important goal of prophylactic or therapeutic vaccine development for human monocytic ehrlichiosis, and perhaps related rickettsial diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Gary Winslow, Wadsworth Center, 120 New Scotland Avenue, Albany, NY 12208. E-mail address: gary.winslow{at}wadsworth.org
3 Abbreviations used in this paper: OMP, outer membrane protein; HVR, hypervariable region; MIWD, mean integrated weight difference; RU, resonance unit.
Received for publication January 29, 2002. Accepted for publication May 29, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-stimulated murine macrophage-like J774.16 cells. J. Leukocyte Biol. 57:657.[Abstract]
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A. Casadevall Antibody-Mediated Immunity against Intracellular Pathogens: Two-Dimensional Thinking Comes Full Circle Infect. Immun., August 1, 2003; 71(8): 4225 - 4228. [Full Text] [PDF]
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J. S.-y. Li and G. M. Winslow Survival, Replication, and Antibody Susceptibility of Ehrlichia chaffeensis outside of Host Cells Infect. Immun., August 1, 2003; 71(8): 4229 - 4237. [Abstract] [Full Text] [PDF]
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 A. D. Loftis, R. F. Massung, and M. L. Levin Quantitative Real-Time PCR Assay for Detection of Ehrlichia chaffeensis J. Clin. Microbiol., August 1, 2003; 41(8): 3870 - 3872. [Abstract] [Full Text] [PDF]
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J. W. McBride, R. E. Corstvet, S. D. Gaunt, C. Boudreaux, T. Guedry, and D. H. Walker Kinetics of Antibody Response to Ehrlichia canis Immunoreactive Proteins Infect. Immun., May 1, 2003; 71(5): 2516 - 2524. [Abstract] [Full Text] [PDF]
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), beginning day 7
postinfection, and at weekly intervals thereafter. The mice were
sacrificed on day 28 postinfection, at which time the PBS-injected mice
were judged to be moribund. Samples of liver, spleen, and peritoneal
exudate from single mice, and pooled peripheral blood from each group
of mice were analyzed using a real-time quantitative PCR assay for
E. chaffeensis 16S rDNA. The copy number of E.
chaffeensis 16S rDNA was enumerated in 5 ng total DNA extracted
from each sample. These correspond to the following bacterial loads in
each tissue from PBS-treated mice, 2.1 x 109 (liver),
1.5 x 109 (spleen), 2.6 x 107
(peritoneal exudate), and 8.3 x 107 (blood/ml); and
Ec56.5-treated mice, 6.0 x 105 (liver), 8.7 x
106 (spleen), 6.9 x 104 (peritoneal
exudate), and 1.1 x 106 (blood/ml). No PCR
amplification product was detected in the control reactions containing
either no template or equivalent amounts of genomic DNA obtained from
uninfected animals. It was previously demonstrated that isotype-matched
irrelevant control Abs had no effect on bacterial load
(
