No evidence of Borrelia in cutaneous infiltrates of B-cell lymphomas with a highly sensitive, semi-nested real-time polymerase chain reaction targeting the 5S-23S intergenic spacer region.

BACKGROUND: The role of Borrelia in the development of skin lymphomas has been under discussion for decades. A similar association has been shown for Helicobacter pylori and gastric lymphomas (MALT type). Nevertheless, few molecular studies investigated Borrelia in skin lymphomas and the results are controversial. METHODS: We analysed 46 formalin-fixed, paraffin-embedded skin specimens of clincopathologically confirmed B-cell lymphomas (15 marginal zone lymphomas; 20 follicular lymphomas; three diffuse large B-cell lymphomas; eight secondary cutaneous infiltrates) taken from 36 patients from Northern Germany, an endemic area for Borrelia. Fifteen pseudolymphomatous lesions of cutaneous Borreliosis served as the control. Both groups were examined with a real-time (rt) PCR and a semi-nested PCR targeting the 5S-23S intergenic spacer region (IGS). A multiplex PCR was used to investigate B-cell clonality in all lymphomatous infiltrates (Biomed Primers). RESULTS: With both assays no Borrelia burgdorferi-specific DNA was identified in any of the B-cell lymphomas, while all 15 Borreliosis specimens gave a positive PCR result in the semi-nested PCR protocol, 12 were also positive in the rt PCR (P < 0.01). All B-cell lymphomas showed monoclonal IgH-Rearrangement. Analysis of cutaneous B-cell lymphomas from available studies including ours (n = 334) reveals an odds ratio <1. CONCLUSION: While some previous studies suggested an association between B. burgdorferi and the development of cutaneous B-cell lymphomas in endemic areas, we were unable to confirm this in our patients, despite a highly sensitive Borrelia PCR assay. Our results including meta-analysis of previous studies question the need for antibiotic therapy in patients with cutaneous B-cell lymphomas.

Combining genome-wide association study and FST -based approaches to identify targets of Borrelia-mediated selection in natural rodent hosts.

Recent advances in high-throughput sequencing technologies provide opportunities to gain novel insights into the genetic basis of phenotypic trait variation. Yet to date, progress in our understanding of genotype-phenotype associations in nonmodel organisms in general and natural vertebrate populations in particular has been hampered by small sample sizes typically available for wildlife populations and a resulting lack of statistical power, as well as a limited ability to control for false-positive signals. Here we propose to combine a genome-wide association study (GWAS) and FST -based approach with population-level replication to partly overcome these limitations. We present a case study in which we used this approach in combination with genotyping-by-sequencing (GBS) single nucleotide polymorphism (SNP) data to identify genomic regions associated with Borrelia afzelii resistance or susceptibility in the natural rodent host of this Lyme disease-causing spirochete, the bank vole (Myodes glareolus). Using this combined approach we identified four consensus SNPs located in exonic regions of the genes Slc26a4, Tns3, Wscd1 and Espnl, which were significantly associated with the voles' Borrelia infectious status within and across populations. Functional links between host responses to bacterial infections and most of these genes have previously been demonstrated in other rodent systems, making them promising new candidates for the study of evolutionary host responses to Borrelia emergence. Our approach is applicable to other systems and may facilitate the identification of genetic variants underlying disease resistance or susceptibility, as well as other ecologically relevant traits, in wildlife populations.

Genomes, expression profiles, and diversity of mitochondria of the White-footed Deermouse Peromyscus leucopus, reservoir of Lyme disease and other zoonoses.

The cricetine rodents Peromyscus leucopus and P. maniculatus are key reservoirs for several zoonotic diseases in North America. We determined the complete circular mitochondrial genome sequences of representatives of 3 different stock colonies of P. leucopus, one stock colony of P. maniculatus and two wild populations of P. leucopus. The genomes were syntenic with that of the murids Mus musculus and Rattus norvegicus. Phylogenetic analysis confirmed that these two Peromyscus species are sister taxa in a clade with P. polionotus and also uncovered a distinction between P. leucopus populations in the eastern and the central United States. In one P. leucopus lineage four extended regions of mitochondrial pseudogenes were identified in the nuclear genome. RNA-seq analysis revealed transcription of the entire genome and differences from controls in the expression profiles of mitochondrial genes in the blood, but not in liver or brain, of animals infected with the zoonotic pathogen Borrelia hermsii. PCR and sequencing of the D-loop of the mitochondrion identified 32 different haplotypes among 118 wild P. leucopus at a Connecticut field site. These findings help to further establish P. leucopus as a model organism for studies of emerging infectious diseases, ecology, and in other disciplines.

Borrelia-primed and -infected mice deficient of interleukin-17 develop arthritis after neutralization of gamma-interferon.

The immune mechanisms responsible for development of Lyme arthritis are partially understood with interleukin-17 (IL-17) and gamma-interferon (IFN-γ) playing a generally accepted role. Elevated levels of IL-17 and/or IFN-γ have been reported in samples from human Lyme arthritis patients and experimental mice. In addition, IL-17 and IFN-γ have been implicated in the onset of arthritis in Borrelia-primed and -infected C57BL/6 mice. Recently, we showed that IL-17-deficient mice developed swelling and histopathological changes consistent with arthritis in the presence of high levels of IFN-γ. We hypothesized that neutralization of IFN-γ in IL-17-deficient mice would inhibit Borrelia-induced arthritis. Our results, however, showed that swelling of the hind paws and histopathological changes of arthritis did not differ between Borrelia-primed and -infected IL-17-deficient and wild-type mice with or without neutralization of IFN-γ. We also found higher levels of tumor necrosis factor alpha (TNF-α) and IL-6 in the popliteal lymph node cells of Borrelia-primed and -infected IL-17-deficient mice after neutralization of IFN-γ. These results suggest that multiple cytokines interact in the development of Borrelia-induced arthritis.

No Evidence that Infection Alters Global Recombination Rate in House Mice.

Recombination rate is a complex trait, with genetic and environmental factors shaping observed patterns of variation. Although recent studies have begun to unravel the genetic basis of recombination rate differences between organisms, less attention has focused on the environmental determinants of crossover rates. Here, we test the effect of one ubiquitous environmental pressure-bacterial infection-on global recombination frequency in mammals. We applied MLH1 mapping to assay global crossover rates in male mice infected with the pathogenic bacterium Borrelia burgdorferi, the causative agent of Lyme Disease, and uninfected control animals. Despite ample statistical power to identify biologically relevant differences between infected and uninfected animals, we find no evidence for a global recombination rate response to bacterial infection. Moreover, broad-scale patterns of crossover distribution, including the number of achiasmate bivalents, are not affected by infection status. Although pathogen exposure can plastically increase recombination in some species, our findings suggest that recombination rates in house mice may be resilient to at least some forms of infection stress. This negative result motivates future experiments with alternative house mouse pathogens to evaluate the generality of this conclusion.

A Coding Variant of ANO10, Affecting Volume Regulation of Macrophages, Is Associated with Borrelia Seropositivity.

In a first genome-wide association study (GWAS) approach to anti-Borrelia seropositivity, we identified two significant single nucleotide polymorphisms (SNPs) (rs17850869, P = 4.17E-09; rs41289586, P = 7.18E-08). Both markers, located on chromosomes 16 and 3, respectively, are within or close to genes previously connected to spinocerebellar ataxia. The risk SNP rs41289586 represents a missense variant (R263H) of anoctamin 10 (ANO10), a member of a protein family encoding Cl(-) channels and phospholipid scramblases. ANO10 augments volume-regulated Cl(-) currents (IHypo) in Xenopus oocytes, HEK293 cells, lymphocytes and macrophages and controls volume regulation by enhancing regulatory volume decrease (RVD). ANO10 supports migration of macrophages and phagocytosis of spirochetes. The R263H variant is inhibitory on IHypo, RVD and intracellular Ca(2+) signals, which may delay spirochete clearance, thereby sensitizing adaptive immunity. Our data demonstrate for the first time that ANO10 has a central role in innate immune defense against Borrelia infection.

Aromatic cluster mutations produce focal modulations of ß-sheet structure.

Site-directed mutagenesis is a powerful tool for altering the structure and function of proteins in a focused manner. Here, we examined how a model ß-sheet protein could be tuned by mutation of numerous surface-exposed residues to aromatic amino acids. We designed these aromatic side chain "clusters" at highly solvent-exposed positions in the flat, single-layer ß-sheet of Borrelia outer surface protein A (OspA). This unusual ß-sheet scaffold allows us to interrogate the effects of these mutations in the context of well-defined structure but in the absence of the strong scaffolding effects of globular protein architecture. We anticipated that the introduction of a cluster of aromatic amino acid residues on the ß-sheet surface would result in large conformational changes and/or stabilization and thereby provide new means of controlling the properties of ß-sheets. Surprisingly, X-ray crystal structures revealed that the introduction of aromatic clusters produced only subtle conformational changes in the OspA ß-sheet. Additionally, despite burying a large degree of hydrophobic surface area, the aromatic cluster mutants were slightly less stable than the wild-type scaffold. These results thereby demonstrate that the introduction of aromatic cluster mutations can serve as a means for subtly modulating ß-sheet conformation in protein design.

Elevated carbon monoxide in the exhaled breath of mice during a systemic bacterial infection.

Blood is the specimen of choice for most laboratory tests for diagnosis and disease monitoring. Sampling exhaled breath is a noninvasive alternative to phlebotomy and has the potential for real-time monitoring at the bedside. Improved instrumentation has advanced breath analysis for several gaseous compounds from humans. However, application to small animal models of diseases and physiology has been limited. To extend breath analysis to mice, we crafted a means for collecting nose-only breath samples from groups and individual animals who were awake. Samples were subjected to gas chromatography and mass spectrometry procedures developed for highly sensitive analysis of trace volatile organic compounds (VOCs) in the atmosphere. We evaluated the system with experimental systemic infections of severe combined immunodeficiency Mus musculus with the bacterium Borrelia hermsii. Infected mice developed bacterial densities of ∼10(7) per ml of blood by day 4 or 5 and in comparison to uninfected controls had hepatosplenomegaly and elevations of both inflammatory and anti-inflammatory cytokines. While 12 samples from individual infected mice on days 4 and 5 and 6 samples from uninfected mice did not significantly differ for 72 different VOCs, carbon monoxide (CO) was elevated in samples from infected mice, with a mean (95% confidence limits) effect size of 4.2 (2.8-5.6), when differences in CO2 in the breath were taken into account. Normalized CO values declined to the uninfected range after one day of treatment with the antibiotic ceftriaxone. Strongly correlated with CO in the breath were levels of heme oxygenase-1 protein in serum and HMOX1 transcripts in whole blood. These results (i) provide further evidence of the informativeness of CO concentration in the exhaled breath during systemic infection and inflammation, and (ii) encourage evaluation of this noninvasive analytic approach in other various other rodent models of infection and for utility in clinical management.

Borrelia burgdorferi linear plasmid 28-3 confers a selective advantage in an experimental mouse-tick infection model.

Borrelia burgdorferi, the bacterium that causes Lyme disease, has a unique segmented genome consisting of numerous linear and circular plasmids and a linear chromosome. Many of these genetic elements have been found to encode factors critical for B. burgdorferi to complete the infectious cycle. However, several plasmids remain poorly characterized, and their roles during infection with B. burgdorferi have not been elucidated. To more fully characterize the role of one of the four 28-kb linear plasmids, lp28-3, we generated strains specifically lacking lp28-3 and assayed the contribution of genes carried by lp28-3 to B. burgdorferi infection. We found that lp28-3 does not carry any genes that are strictly required for infection of a mouse or tick and that lp28-3-deficient spirochetes are competent at causing a disseminated infection. Interestingly, spirochetes containing lp28-3 were at a selective advantage compared to lp28-3-deficient spirochetes when coinjected into a mouse, and this advantage was reflected in the population of spirochetes acquired by feeding ticks. Our data demonstrate that genes carried by lp28-3, although not essential, contribute to the fitness of B. burgdorferi during infection.

Borrelia hermsii acquisition order in superinfected ticks determines transmission efficiency.

Multilocus sequence typing of Borrelia hermsii isolates reveals its divergence into two major genomic groups (GG), but no differences in transmission efficiency or host pathogenicity are associated with these genotypes. To compare GGI and GGII in the tick-host infection cycle, we first determined if spirochetes from the two groups could superinfect the tick vector Ornithodoros hermsi. We infected mice with isolates from each group and fed ticks sequentially on these mice. We then fed the infected ticks on naive mice and measured GGI and GGII spirochete densities in vector and host, using quantitative PCR of genotype-specific chromosomal DNA sequences. Sequential feedings resulted in dual tick infections, showing that GGI or GGII primary acquisition did not block superinfection by a secondary agent. On transmission to naive mice at short intervals after acquisition, ticks with primary GGI and secondary GGII spirochete infections caused mixed GGI and GGII infections in mice. However, ticks with primary GGII and secondary GGI spirochete infections caused only GGII infections with all isolate pairs examined. At longer intervals after acquisition, the exclusion of GGI by GGII spirochetes declined and cotransmission predominated. We then examined GGI and GGII spirochetemia in mice following single inoculation and coinoculation by needle and found that GGI spirochete densities were reduced on multiple days when coinoculated with GGII. These findings indicate that dual GGI-GGII spirochete infections can persist in ticks and that transmission to a vertebrate host is dependent on the order of tick acquisition and the interval between acquisition and transmission events.

Acrodermatitis chronica atrophicans with pseudolymphomatous infiltrates.

In this study, we describe the clinicopathologic features of pseudolymphomatous infiltrates found within lesions of acrodermatitis chronica atrophicans (ACA). We studied 11 patients (10 females, 1 male, age range 60-88 years). The diagnosis of ACA in all cases was confirmed by clinicopathologic correlation and positive serology for Borrelia. Histopathologic examination revealed prominent, pseudolymphomatous inflammatory cell infiltrates in all cases, with 2 distinct patterns. Eight of 11 cases showed a band-like lymphocytic infiltrate, exocytosis of lymphocytes and a fibrotic papillary dermis, similar to features seen in mycosis fungoides. The other 3 cases showed dense, nodular-diffuse dermal infiltrates with many plasma cells and without germinal centers. The plasma cells expressed both kappa and lambda immunoglobulin light chains with a polyclonal pattern in all 3 cases. In conclusion, ACA may present with pseudolymphomatous infiltrates showing both a T-cell and, less frequently, a B-cell pattern. These lesions need to be distinguished from a cutaneous lymphoma. In the context of the knowledge of Borrelia-associated cutaneous lymphomas, follow-up seems advisable in these cases.

B1b lymphocyte-derived antibodies control Borrelia hermsii independent of Fcα/µ receptor and in the absence of host cell contact.

The critical role of IgM in controlling pathogen burden has been demonstrated in a variety of infection models. In the murine model of Borrelia hermsii infection, IgM is necessary and sufficient for the rapid clearance of bacteremia. Convalescent, but not naïve, B1b cells generate a specific IgM response against B. hermsii, but the mechanism of IgM-mediated protection is unknown. Here, we show that neither Fcα/µR, a high-affinity receptor for IgM, nor IgM-dependent complement activation is required for controlling B. hermsii. Bacteria in diffusion chambers with a pore size impermeable to cells were killed when diffusion chambers were implanted into either convalescent or passively immunized mice. Furthermore, adoptively transferred convalescent B1b cells in Rag1(-/-) mice produced specific IgM that also cleared B. hermsii in diffusion chambers independent of complement. These results demonstrate that IgM-mediated clearance of B. hermsii does not require opsonophagocytosis and indicate that a mechanism for in vivo B1b cell-mediated protection is through the generation of bactericidal IgM.

BBA52 facilitates Borrelia burgdorferi transmission from feeding ticks to murine hosts.

Borrelia burgdorferi, the pathogen of Lyme borreliosis, persists in nature through a tick-rodent transmission cycle. A selective assessment of the microbial transcriptome, limited to gene-encoding putative membrane proteins, reveals that bba52 transcription in vivo is strictly confined to the vector-specific portion of the microbial life cycle, with the highest levels of expression noted in feeding ticks and with swift down-regulation noted in mice. bba52 deletion did not affect murine disease as assessed by the genesis of arthritis and carditis or long-term persistence of pathogens in mice or ticks. However, bba52 deficiency did impair microbial transitions between hosts and vector, defects that could be fully rescued when bba52 expression was genetically restored to the original genomic locus. These studies establish that BBA52 facilitates vector-host transitions by the pathogen and therefore is a potential antigenic target for interference with transmission of B. burgdorferi from ticks to mammalian hosts.

Borrelia in granuloma annulare, morphea and lichen sclerosus: a PCR-based study and review of the literature.

BACKGROUND: Morphea, granuloma annulare (GA) and lichen sclerosus et atrophicans (LSA) have also been suggested to be linked to Borrelia infection. Previous studies based on serologic data or detection of Borrelia by immunohistochemistry and polymerase chain reaction (PCR) reported contradictory results. Thus, we examined skin biopsies of morphea, GA and LSA by PCR to assess the prevalence of Borrelia DNA in an endemic area and to compare our results with data in the literature. METHODS: Amplification of DNA sequences of Borrelia burgdorferi sensu lato by nested PCR from formalin-fixed and paraffin-embedded skin biopsies of morphea, GA and LSA, followed by automated sequencing of amplification products. PCR-based studies on Borrelia species in these disorders published until July 2009 were retrieved by a literature search. RESULTS: Borrelia DNA was detected in 3 of 112 skin biopsies (2.7%) including one of 49 morphea biopsies (2.0%), one of 48 GA biopsies (2.1%) and one of 15 LSA biopsies (6.6%). Amplification products belonged to B. burgdorferi sensu stricto in two cases available for sequence analysis. CONCLUSIONS: The results of our and most of other PCR-based studies do not argue for a significant association of B. burgdorferi sensu lato with morphea, GA, LSA.

CD14 signaling restrains chronic inflammation through induction of p38-MAPK/SOCS-dependent tolerance.

Current thinking emphasizes the primacy of CD14 in facilitating recognition of microbes by certain TLRs to initiate pro-inflammatory signaling events and the importance of p38-MAPK in augmenting such responses. Herein, this paradigm is challenged by demonstrating that recognition of live Borrelia burgdorferi not only triggers an inflammatory response in the absence of CD14, but one that is, in part, a consequence of altered PI3K/AKT/p38-MAPK signaling and impaired negative regulation of TLR2. CD14 deficiency results in increased localization of PI3K to lipid rafts, hyperphosphorylation of AKT, and reduced activation of p38. Such aberrant signaling leads to decreased negative regulation by SOCS1, SOCS3, and CIS, thereby compromising the induction of tolerance in macrophages and engendering more severe and persistent inflammatory responses to B. burgdorferi. Importantly, these altered signaling events and the higher cytokine production observed can be mimicked through shRNA and pharmacological inhibition of p38 activity in CD14-expressing macrophages. Perturbation of this CD14/p38-MAPK-dependent immune regulation may underlie development of infectious chronic inflammatory syndromes.

IL-10 helps control pathogen load during high-level bacteremia.

During relapsing fever borreliosis, a high pathogen load in the blood occurs at times of peak bacteremia. Specific IgM Abs are responsible for spirochetal clearance so in absence of B cells there is persistent high-level bacteremia. Previously, we showed that B cell-deficient mice persistently infected with Borrelia turicatae produce high levels of IL-10 and that exogenous IL-10 reduces bacteremia. This suggested that IL-10 helps reduce bacteremia at times of high pathogen load by a B cell-independent mechanism, most likely involving innate immunity. To investigate this possibility, we compared B. turicatae infection in RAG2/IL-10(-/-) and RAG2(-/-) mice. The results showed that IL-10 deficiency resulted in significantly higher bacteremia, higher TNF levels, and early mortality. Examination of the spleen and peripheral blood showed markedly increased apoptosis of immune cells in infected RAG2/IL-10(-/-) mice. Neutralization of TNF reduced apoptosis of leukocytes and splenocytes, increased production of IFN-gamma by NK cells, increased phagocytosis in the spleen, decreased spirochetemia, and rescued mice from early death. Our results indicate that at times of high pathogen load, as during peak bacteremia in relapsing fever borreliosis, IL-10 protects innate immune cells from apoptosis via inhibition of TNF resulting in improved pathogen control.

A differential role for BB0365 in the persistence of Borrelia burgdorferi in mice and ticks.

Borrelia burgdorferi, the etiologic agent of Lyme disease, persists in both an arthropod vector and vertebrate hosts, usually wild rodents. Analysis of the B. burgdorferi transcriptome in vivo indicates that the bb0365 gene is markedly induced as spirochetes enter the feeding ticks from infected mice. To understand the importance of the bb0365 gene product in the spirochete life cycle, we inactivated this gene in an infectious isolate of B. burgdorferi B31. BB0365-deficient spirochetes were fully pathogenic in mice and survived in diverse murine tissues. When naive ticks engorged on spirochete-infected mice, the B. burgdorferi bb0365 mutant entered ticks but had a markedly decreased survival rate compared with wild type B. burgdorferi. BB0365 therefore is not necessary for B. burgdorferi persistence in the vertebrate host but is required for survival of the Lyme disease agent within the feeding arthropod vector, and strategies for interfering with this gene may potentially interrupt the B. burgdorferi life cycle.

MyD88- and Bruton's tyrosine kinase-mediated signals are essential for T cell-independent pathogen-specific IgM responses.

Bacteremia is one of the leading causes of death by infectious disease. To understand the immune mechanisms required for the rapid control of bacteremia, we studied Borrelia hermsii, a bacterial pathogen that colonizes the blood stream of humans and rodents to an extremely high density. A T cell-independent IgM response is essential and sufficient for controlling B. hermsii bacteremia. Mice deficient in Bruton's tyrosine kinase (Btk), despite their known defect in BCR signaling, generated B. hermsii-specific IgM and resolved bacteremia, suggesting that an alternative activation or costimulatory pathway remained functional for T cell-independent B cells in Btk(-/-) mice. B. hermsii contains putative ligands for TLRs, and we found that mice deficient in TLR1, TLR2, or the TLR adaptor MyD88 generated anti-B. hermsii IgM with delayed kinetics and suffered more severe episodes of bacteremia. In striking contrast to the anti-B. hermsii IgM response in mice deficient only in Btk, mice deficient in both Btk and MyD88 were entirely incapable of generating B. hermsii-specific Ab or resolving bacteremia. The response to a T cell-dependent model Ag was unaffected in Btk(-/-) x MyD88(-/-) mice. These results suggest that MyD88 specifically promotes T cell-independent BCR signaling and that, in the absence of Btk, this TLR-mediated stimulation is a required component of this signal.

Identification of a TLR-independent pathway for Borrelia burgdorferi-induced expression of matrix metalloproteinases and inflammatory mediators through binding to integrin alpha 3 beta 1.

Borrelia burgdorferi stimulates a robust inflammatory response at sites of localization. Binding of borrelial lipoproteins to TLR-2 is one pathway important in the host response to B. burgdorferi. However, while TLR-2 is clearly important in control of infection, inflammation is actually worsened in the absence of TLR-2 or the shared TLR adapter molecule, MyD88, suggesting that there are alternative pathways regulating inflammation. Integrins are cell surface receptors that play an important role in cell to cell communications and that can activate inflammatory signaling pathways. In this study, we report for the first time that B. burgdorferi binds to integrin alpha(3)beta(1) and that binding of B. burgdorferi to this integrin results in induction of proinflammatory cytokines, chemokines, and end-effector molecules such as matrix metalloproteinases in primary human chondrocyte cells. Expression of these same molecules is not affected by the absence of MyD88 in murine articular cartilage, suggesting that the two pathways act independently in activating host inflammatory responses to B. burgdorferi. B. burgdorferi-induced alpha(3) signaling is mediated by JNK, but not p38 MAPK. In summary, we have identified a new host receptor for B. burgdorferi, integrin alpha(3)beta(1); binding of B. burgdorferi to integrin alpha(3)beta(1) results in the release of inflammatory mediators and is proposed as a TLR-independent pathway for activation of the innate immune response by the organism.

Infection-induced marginal zone B cell production of Borrelia hermsii-specific antibody is impaired in the absence of CD1d.

Ab that arise in the absence of T cell help are a critical host defense against infection with the spirochetes Borrelia burgdorferi and Borrelia hermsii. We have previously shown that CD1d-deficient (CD1d(-/-)) mice have impaired resistance to infection with B. burgdorferi. In mice, CD1d expression is highest on marginal zone B (MZB) cells, which produce Ab to blood-borne Ag. In this study we examined MZB cell activation and Ab production in mice infected with B. hermsii, which achieve high levels of bacteremia. We show by flow cytometry that MZB cells associate with B. hermsii and up-regulate the activation markers syndecan I and B7.1 within 16 h of infection. By 24 h, MZB cells secrete B. hermsii-specific IgM, coinciding with the loss of activation marker expression and the reduction in spirochete burden. In contrast, MZB cells from CD1d(-/-) mice remain activated for at least 96 h of infection, but produce only minimal B. hermsii-specific IgM in vivo and ex vivo; pathogen burden in the blood also remains elevated. Wild-type mice depleted of MZB cells using mAb to LFA-1 and alpha(4)beta(1) integrin have reduced serum levels of B. hermsii-specific IgM and increased pathogen burden, similar to B. hermsii-infected CD1d(-/-) mice. Passive transfer of immune mouse serum, but not naive mouse serum, into infected CD1d(-/-) mice leads to down-regulation of activation markers and clearance of B. hermsii from the MZB cells. These results demonstrate that blood-borne spirochetes activate MZB cells to produce pathogen-specific IgM and reveal a role for CD1d in this process.