Borrelia burgdorferi Infection-Induced Persistent IgM Secretion Controls Bacteremia, but Not Bacterial Dissemination or Tissue Burden.

Infection with Borrelia burgdorferi causes Lyme disease in humans. In small rodents, the natural reservoir species of this spirochete, infections lead to only modest disease manifestations, despite causing persistence infection. Although B cell responses are central for controlling bacterial tissue burden and disease manifestations, they lack classical aspects of T-dependent responses, such as sustained IgG affinity maturation and longevity, corresponding with a rapid collapse of germinal centers. Instead, the Ab response is characterized by strong and ongoing secretion of IgM, whose origins and impact on protective immunity to B. burgdorferi remain unknown. In this article, we demonstrate that B. burgdorferi infection-induced IgM in mice was produced continuously, mainly by conventional B, not B-1 cells, in a T-independent manner. Although IgM was passively protective and restricted early bacteremia, its production had no effects on bacterial dissemination into solid tissues, nor did it affect Borrelia tissue burden. The latter was controlled by the induction of bactericidal IgG, as shown comparing infections in wild type mice with those of mice lacking exclusively secreted IgM-/-, all class-switched Abs via deletion of aicda (AID-/-), and all secreted Abs (secreted IgM-/- × AID-/-). Consistent with the notion that B. burgdorferi infection drives production of IgM over more tissue-penetrable IgG, we demonstrated increased short- and long-term IgM Ab responses also to a coadministered, unrelated Ag. Thus, the continued production of IgM may explain the absence of B. burgdorferi in the blood.

MyD88- and TRIF-independent induction of type I interferon drives naive B cell accumulation but not loss of lymph node architecture in Lyme disease.

Rapidly after infection, live Borrelia burgdorferi, the causative agent of Lyme disease, is found within lymph nodes, causing rapid and strong tissue enlargement, a loss of demarcation between B cell follicles and T cell zones, and an unusually large accumulation of B cells. We sought to explore the mechanisms underlying these changes, as lymph tissue disruption could be detrimental for the development of robust Borrelia-specific immunity. A time course study demonstrated that the loss of the normal lymph node structure was a distinct process that preceded the strong increases in B cells at the site. The selective increases in B cell frequencies were due not to proliferation but rather to cytokine-mediated repositioning of B cells to the lymph nodes, as shown with various gene-targeted and bone marrow irradiation chimeras. These studies demonstrated that B. burgdorferi infection induced type I interferon receptor (IFNR) signaling in lymph nodes in a MyD88- and TRIF-independent manner and that type I IFNR indirect signaling was required for the excessive increases of naive B cells at those sites. It did not, however, drive the observed histopathological changes, which occurred independently also from major shifts in the lymphocyte-homing chemokines, CXCL12, CXCL13, and CCL19/21, as shown by quantitative reverse transcription-PCR (qRT-PCR), flow cytometry, and transwell migration experiments. Thus, B. burgdorferi infection drives the production of type I IFN in lymph nodes and in so doing strongly alters the cellular composition of the lymph nodes, with potential detrimental effects for the development of robust Borrelia-specific immunity.

Vaccination against a virus-encoded cytokine significantly restricts viral challenge.

Identification of immune correlates of protection for viral vaccines is complicated by multiple factors, but there is general consensus on the importance of antibodies that neutralize viral attachment to susceptible cells. Development of new viral vaccines has mostly followed this neutralizing antibody paradigm, but as a recent clinical trial of human cytomegalovirus (HCMV) vaccination demonstrated, this singular approach can yield limited protective efficacy. Since HCMV devotes >50% of its coding capacity to proteins that modulate host immunity, it is hypothesized that expansion of vaccine targets to include this part of the viral proteome will disrupt viral natural history. HCMV and rhesus cytomegalovirus (RhCMV) each encode an ortholog to the cellular interleukin-10 (cIL-10) cytokine: cmvIL-10 and rhcmvIL10, respectively. Despite extensive sequence divergence from their host's cIL-10, each viral IL-10 retains nearly identical functionality to cIL-10. Uninfected rhesus macaques were immunized with engineered, nonfunctional rhcmvIL-10 variants, which were constructed by site-directed mutagenesis to abolish binding to the cIL-10 receptor. Vaccinees developed antibodies that neutralized rhcmvIL-10 function with no cross-neutralization of cIL-10. Following subcutaneous RhCMV challenge, the vaccinees exhibited both reduced RhCMV replication locally at the inoculation site and systemically and significantly reduced RhCMV shedding in bodily fluids compared to controls. Attenuation of RhCMV infection by rhcmvIL-10 vaccination argues that neutralization of viral immunomodulation may be a new vaccine paradigm for HCMV by expanding potential vaccine targets.

Delays and diversions mark the development of B cell responses to Borrelia burgdorferi infection.

B cell responses modulate disease during infection with Borrelia burgdorferi, the causative agent of Lyme disease, but are unable to clear the infection. Previous studies have demonstrated that B. burgdorferi infection induces predominantly T-independent B cell responses, potentially explaining some of these findings. However, others have shown effects of T cells on the isotype profile and the magnitude of the B. burgdorferi-specific Abs. This study aimed to further investigate the humoral response to B. burgdorferi and its degree of T cell dependence, with the ultimate goal of elucidating the mechanisms underlying the failure of effective immunity to this emerging infectious disease agent. Our study identifies distinct stages in the B cell response using a mouse model, all marked by the generation of unusually strong and persistent T-dependent and T-independent IgM Abs. The initial phase is dominated by a strong T-independent accumulation of B cells in lymph nodes and the induction of specific Abs in the absence of germinal centers. A second phase begins around week 2.5 to 3, in which relatively short-lived germinal centers develop in lymph nodes, despite a lymph node architecture that lacks clearly demarcated T and B cell zones. This response failed, however, to generate appreciable numbers of long-lived bone marrow plasma cells. Finally, there is a slow accumulation of long-lived Ab-secreting plasma cells in bone marrow, reflected by a strong but ultimately ineffective serum Ab response. Overall, the study indicates that B. burgdorferi might evade B cell immunity by interfering with its response kinetics and quality.

The early dissemination defect attributed to disruption of decorin-binding proteins is abolished in chronic murine Lyme borreliosis.

The laboratory mouse model of Lyme disease has revealed that Borrelia burgdorferi differentially expresses numerous outer surface proteins that influence different stages of infection (tick-borne transmission, tissue colonization, dissemination, persistence, and tick acquisition). Deletion of two such outer surface proteins, decorin-binding proteins A and B (DbpA/B), has been documented to decrease infectivity, impede early dissemination, and, possibly, prevent persistence. In this study, DbpA/B-deficient spirochetes were confirmed to exhibit an early dissemination defect in immunocompetent, but not immunodeficient, mice, and the defect was found to resolve with chronicity. Development of disease (arthritis and carditis) was attenuated only in the early stage of infection with DbpA/B-deficient spirochetes in both types of mice. Persistence of the DbpA/B-deficient spirochetes occurred in both immunocompetent and immunodeficient mice in a manner indistinguishable from that of wild-type spirochetes. Dissemination through the lymphatic system was evaluated as an underlying mechanism for the early dissemination defect. At 12 h, 3 days, 7 days, and 14 days postinoculation, DbpA/B-deficient spirochetes were significantly less prevalent and in lower numbers in lymph nodes than wild-type spirochetes. However, in immunodeficient mice, deficiency of DbpA/B did not significantly decrease the prevalence or spirochete numbers in lymph nodes. Complementation of DbpA/B restored a wild-type phenotype. Thus, the results indicated that deficiency of DbpA/B allows the acquired immune response to restrict early dissemination of spirochetes, which appears to be at least partially mediated through the lymphatic system.

Dynamics of connective-tissue localization during chronic Borrelia burgdorferi infection.

The etiologic agent of Lyme disease, Borrelia burgdorferi, localizes preferentially in the extracellular matrix during persistence. In chronically infected laboratory mice, there is a direct association between B. burgdorferi and the proteoglycan decorin, which suggests that decorin has a role in defining protective niches for persistent spirochetes. In this study, the tissue colocalization of B. burgdorferi with decorin and the dynamics of borrelial decorin tropism were evaluated during chronic infection. Spirochetes were found to colocalize absolutely with decorin, but not collagen I in chronically infected immunocompetent C3H mice. Passive immunization of infected C3H-scid mice with B. burgdorferi-specific immune serum resulted in the localization of spirochetes in decorin-rich microenvironments, with clearance of spirochetes from decorin-poor microenvironments. In passively immunized C3H-scid mice, tissue spirochete burdens were initially reduced, but increased over time as the B. burgdorferi-specific antibody levels waned. Concurrent repopulation of the previously cleared decorin-poor microenvironments was observed with the rising tissue spirochete burden and declining antibody titer. These findings indicate that the specificity of B. burgdorferi tissue localization during chronic infection is determined by decorin, driven by the borrelia-specific antibody response, and fluctuates with the antibody response.

Lymphoadenopathy during lyme borreliosis is caused by spirochete migration-induced specific B cell activation.

Lymphadenopathy is a hallmark of acute infection with Borrelia burgdorferi, a tick-borne spirochete and causative agent of Lyme borreliosis, but the underlying causes and the functional consequences of this lymph node enlargement have not been revealed. The present study demonstrates that extracellular, live spirochetes accumulate in the cortical areas of lymph nodes following infection of mice with either host-adapted, or tick-borne B. burgdorferi and that they, but not inactivated spirochetes, drive the lymphadenopathy. The ensuing lymph node response is characterized by strong, rapid extrafollicular B cell proliferation and differentiation to plasma cells, as assessed by immunohistochemistry, flow cytometry and ELISPOT analysis, while germinal center reactions were not consistently observed. The extrafollicular nature of this B cell response and its strongly IgM-skewed isotype profile bear the hallmarks of a T-independent response. The induced B cell response does appear, however, to be largely antigen-specific. Use of a cocktail of recombinant, in vivo-expressed B. burgdorferi-antigens revealed the robust induction of borrelia-specific antibody-secreting cells by ELISPOT. Furthermore, nearly a quarter of hybridomas generated from regional lymph nodes during acute infection showed reactivity against a small number of recombinant Borrelia-antigens. Finally, neither the quality nor the magnitude of the B cell responses was altered in mice lacking the Toll-like receptor adaptor molecule MyD88. Together, these findings suggest a novel evasion strategy for B. burgdorferi: subversion of the quality of a strongly induced, potentially protective borrelia-specific antibody response via B. burdorferi's accumulation in lymph nodes.

Influence of arthritis-related protein (BBF01) on infectivity of Borrelia burgdorferi B31.

BACKGROUND: Lyme borreliosis, caused by tick-borne Borrelia burgdorferi, is a multi-phasic, multi-system disease in humans. Similar to humans, C3H mice develop arthritis and carditis, with resolution and periodic bouts of recurrence over the course of persistent infection. Borrelia burgdorferi arthritis-related protein (Arp/BBF01), a highly conserved protein among B. burgdorferi s.s. isolates, has been shown to be antigenic in humans with Lyme borreliosis, and a target for antibody-mediated disease resolution in the mouse model. RESULTS: A mutant strain of B. burgdorferi s.s. deficient of the arp gene and a complemented version of that mutant were created and examined for phenotypic effects in mice compared to wild-type B. burgdorferi. Deletion of arp did not abolish infectivity, but did result in a higher infectious dose compared to wild-type B. burgdorferi, which was restored by complementation. Spirochete burdens in tissues of C3H-scid mice were lower when infected with the arp mutant, compared to wild-type, but arthritis was equally severe. Spirochete burdens were also lower in C3H mice infected with the arp mutant, but disease was markedly reduced. Ticks that fed upon infected C3H mice were able to acquire infection with both wild-type and arp mutant spirochetes. Arp mutant spirochetes were marginally able to be transmitted to naïve hosts by infected ticks. CONCLUSION: These results indicated that deletion of BBF01/arp did not abrogate, but diminished infectivity and limited spirochete burdens in tissues of both immunocompetent and immunodeficient hosts, and attenuated, but did not abolish the ability of ticks to acquire or transmit infection.

Comparative molecular analyses of Borrelia burgdorferi sensu stricto strains B31 and N40D10/E9 and determination of their pathogenicity.

BACKGROUND: Lyme disease in the United States is caused primarily by B. burgdorferi sensu stricto while other species are also prevalent in Europe. Genetic techniques have identified several chromosomal and plasmid-borne regulatory and virulence factors involved in Lyme pathogenesis. B31 and N40 are two widely studied strains of B. burgdorferi, which belong to two different 16 S-23 S rRNA spacer types (RST) and outer surface protein C (OspC) allelic groups. However, the presence of several known virulence factors in N40 has not been investigated. This is the first comprehensive study that compared these two strains both in vitro and using the mouse model of infection. RESULTS: Phylogenetic analyses predict B31 to be more infectious. However, our studies here indicate that N40D10/E9 is more infectious than the B31 strain at lower doses of inoculation in the susceptible C3H mice. Based-upon a careful analyses of known adhesins of these strains, it is predicted that the absence of a known fibronectin-glycosaminoglycan binding adhesin, bbk32, in the N40 strain could at least partially be responsible for reduction in its binding to Vero cells in vitro. Nevertheless, this difference does not affect the infectivity of N40D10/E9 strain. The genes encoding known regulatory and virulence factors critical for pathogenesis were detected in both strains. Differences in the protein profiles of these B. burgdorferi strains in vitro suggest that the novel, differentially expressed molecules may affect infectivity of B. burgdorferi. Further exacerbation of these molecular differences in vivo could affect the pathogenesis of spirochete strains. CONCLUSION: Based upon the studies here, it can be predicted that N40D10/E9 disseminated infection at lower doses may be enhanced by its lower binding to epithelial cells at the site of inoculation due to the absence of BBK32. We suggest that complete molecular analyses of virulence factors followed by their evaluation using the mouse infection model should form the basis of determining infectivity and pathogenicity of different strains rather than simple phylogenetic group analyses. This study further emphasizes a need to investigate multiple invasive strains of B. burgdorferi to fully appreciate the pathogenic mechanisms that contribute to Lyme disease manifestations.

Ineffectiveness of tigecycline against persistent Borrelia burgdorferi.

The effectiveness of a new first-in-class antibiotic, tigecycline (glycylcycline), was evaluated during the early dissemination (1 week), early immune (3 weeks), or late persistent (4 months) phases of Borrelia burgdorferi infection in C3H mice. Mice were treated with high or low doses of tigecycline, saline (negative-effect controls), or a previously published regimen of ceftriaxone (positive-effect controls). Infection status was assessed at 3 months after treatment by culture, quantitative ospA real-time PCR, and subcutaneous transplantation of joint and heart tissue into SCID mice. Tissues from all saline-treated mice were culture and ospA PCR positive, tissues from all antibiotic-treated mice were culture negative, and some of the tissues from most of the mice treated with antibiotics were ospA PCR positive, although the DNA marker load was markedly decreased compared to that in saline-treated mice. Antibiotic treatment during the early stage of infection appeared to be more effective than treatment that began during later stages of infection. The viability of noncultivable spirochetes in antibiotic-treated mice (demonstrable by PCR) was confirmed by transplantation of tissue allografts from treated mice into SCID mice, with dissemination of spirochetal DNA to multiple recipient tissues, and by xenodiagnosis, including acquisition by ticks, transmission by ticks to SCID mice, and survival through molting into nymphs and then into adults. Furthermore, PCR-positive heart base tissue from antibiotic-treated mice revealed RNA transcription of several B. burgdorferi genes. These results extended previous studies with ceftriaxone, indicating that antibiotic treatment is unable to clear persisting spirochetes, which remain viable and infectious, but are nondividing or slowly dividing.

'One medicine---one pathology': are veterinary and human pathology prepared?

The American Medical Association and the American Veterinary Medical Association have recently approved resolutions supporting 'One Medicine' or 'One Health' that bridge the two professions. The concept is far from novel. Rudolf Virchow, the Father of Modern Pathology, and Sir William Osler, the Father of Modern Medicine, were outspoken advocates of the concept. The concept in its modern iteration was re-articulated in the 1984 edition of Calvin Schwabe's 'Veterinary Medicine and Human Health.' The veterinary and medical pathology professions are steeped in a rich history of 'One Medicine,' but they have paradoxically parted ways, leaving the discipline of pathology poorly positioned to contribute to contemporary science. The time has come for not only scientists but also all pathologists to recognize the value in comparative pathology, the consequences of ignoring the opportunity and, most importantly, the necessity of preparing future generations to meet the challenge inherent in the renewed momentum for 'One Medicine.' The impending glut of new genetically engineered mice creates an urgent need for prepared investigators and pathologists.

Persistence of Borrelia burgdorferi following antibiotic treatment in mice.

The effectiveness of antibiotic treatment was examined in a mouse model of Lyme borreliosis. Mice were treated with ceftriaxone or saline solution for 1 month, commencing during the early (3 weeks) or chronic (4 months) stages of infection with Borrelia burgdorferi. Tissues from mice were tested for infection by culture, PCR, xenodiagnosis, and transplantation of allografts at 1 and 3 months after completion of treatment. In addition, tissues were examined for the presence of spirochetes by immunohistochemistry. In contrast to saline solution-treated mice, mice treated with antibiotic were consistently culture negative, but tissues from some of the mice remained PCR positive, and spirochetes could be visualized in collagen-rich tissues. Furthermore, when some of the antibiotic-treated mice were fed on by Ixodes scapularis ticks (xenodiagnosis), spirochetes were acquired by the ticks, as determined based upon PCR results, and ticks from those cohorts transmitted spirochetes to naïve SCID mice, which became PCR positive but culture negative. Results indicated that following antibiotic treatment, mice remained infected with nondividing but infectious spirochetes, particularly when antibiotic treatment was commenced during the chronic stage of infection.

P26-based serodiagnosis for Bartonella spp. infection in cats.

Bartonella henselae P26 has been identified as an immunodominant antigen expressed during feline infection. We used antisera from cats experimentally infected with B. henselae (n = 6), B. clarridgeiae (n = 4), or B. koehlerae (n = 2) and from a sample of naturally infected cats (B. henselae, n = 34; B. clarridgeiae, n = 1) to evaluate recombinant P26 (rP26) as a serodiagnostic antigen. Immunoblots using antisera from cats infected with B. henselae and B. clarridgeiae reacted strongly with rP26, whereas B. koehlerae antisera did not. A capture ELISA was designed to evaluate the kinetics of rP26 IgG in sera from experimentally infected cats. For B. henselae and B. clarridgeiae antisera, the kinetic profiles of reactivity were similar for rP26 capture ELISA and Bartonella spp. indirect fluorescence assay. However, for B. koehlerae antisera, reactivity in rP26 capture ELISA was consistently low. The serodiagnostic potential of rP26 capture ELISA was evaluated using sera from cats with known Bartonella sp. exposure histories. All 24 (100%) uninfected cats were seronegative, and 33 of 35 (94.3%) cats bacteremic for Bartonella spp. were seropositive. We propose that rP26-based serology can serve as a useful adjunct tool for the diagnosis of feline infection with B. henselae and B. clarridgeiae, but it may not be useful for feline infection with B. koehlerae.

Coinfection with Borrelia burgdorferi sensu stricto and Borrelia garinii alters the course of murine Lyme borreliosis.

Ixodes ricinus ticks and mice can be infected with both Borrelia burgdorferi sensu stricto and Borrelia garinii. The effect of coinfection with these two Borrelia species on the development of murine Lyme borreliosis is unknown. Therefore, we investigated whether coinfection with the nonarthritogenic B. garinii strain PBi and the arthritogenic B. burgdorferi sensu stricto strain B31 alters murine Lyme borreliosis. Mice simultaneously infected with PBi and B31 showed significantly more paw swelling and arthritis, long-standing spirochetemia, and higher numbers of B31 spirochetes than did mice infected with B31 alone. However, the number of PBi spirochetes was significantly lower in coinfected mice than in mice infected with PBi alone. In conclusion, simultaneous infection with B. garinii and B. burgdorferi sensu stricto results in more severe Lyme borreliosis. Moreover, we suggest that competition of the two Borrelia species within the reservoir host could have led to preferential maintenance, and a rising prevalence, of B. burgdorferi sensu stricto in European I. ricinus populations.

Experimental infection of domestic cats with passaged genotype I Bartonella henselae.

The influence of in vitro passage on Bartonella henselae pathogenesis in cats has not been thoroughly evaluated. Our objective was to examine the bacterial kinetics and humoral immune responses in cats experimentally infected with three different in vitro passages of B. henselae F1, a genotype I strain of feline origin. The F1 strain was in vitro passaged 20 and 40 times, and each was inoculated into a group of 5 cats. The kinetics of bacteremia and the feline humoral immune response to bacterial antigens were compared to a previous study involving a group of six cats inoculated with the original F1 strain. Among the three groups of cats, the kinetics of bacteremia profiles and the humoral immune responses to B. henselae lysates were similar. The influence of passage on bacterial membrane proteins was examined. In vitro passage altered the expression of 4/17 (23.5%) bacterial membrane proteins and 6/15 (40%) bacterial membrane antigens. An association between poor seroreactivity to three lysate antigens (15-, 18- and 45kDa), prolonged bacteremia and decreased serum bactericidal activity was noted. Our data show that in vitro passage of B. henselae did not alter the kinetics of bacteremia, including the occurrence of relapsing bacteremia, in experimentally infected cats. This suggests that highly passaged strains may not be suitable for future vaccination studies. Furthermore, in vitro passage results in phenotypic and antigenic changes in the bacterial membrane protein profile, which warrants caution in the interpretation of studies involving passaged B. henselae strains.

Utilizing a TLR5-Adjuvanted Cytomegalovirus as a Lentiviral Vaccine in the Nonhuman Primate Model for AIDS.

Despite tremendous progress in our understanding of human immunodeficiency virus (HIV) natural history and advances in HIV treatment, there is neither an approved vaccine nor a cure for infection. Here, we describe the development and characterization of a novel replicating vaccine vector utilizing Cytomegalovirus (CMV) and a TLR5 adjuvant. After partial truncation of the central, immunodominant hypervariable domain, flagellin (fliC) from Salmonella was cloned downstream of a codon optimized gag gene from simian immunodeficiency virus (SIV) and transiently expressed in telomerized rhesus fibroblast (TeloRF) cells in culture. Lysates generated from these transfected cells induced the tumor necrosis factor alpha (TNF-α), in a mouse macrophage cell line, in a TLR5-dependent manner. The Gag/FliC expression construct was cloned into a bacterial artificial chromosome encoding the rhesus CMV (RhCMV) genome, and infectious RhCMV was generated following transfection of TeloRF cells. This virus stably expressed an SIV Gag/FliC fusion protein through four serial passages. Lysates generated from infected cells induced TNF-α in a TLR5-dependent manner. Western blot analysis of infected cell lysates verified expression of a Gag/FliC fusion protein using a SIV p27 capsid monoclonal antibody. Lastly, rhesus macaques inoculated with this novel RhCMV virus demonstrated increased inflammatory responses at the site of inoculation seven days post-infection when compared to the parental RhCMV. These results demonstrate that an artificially constructed replicating RhCMV expressing an SIV Gag/FliC fusion protein is capable of activating TLR5 in a macrophage cell line in vitro and induction of an altered inflammatory response in vivo. Ongoing animals studies are aimed at determining vaccine efficacy, including subsequent challenge with pathogenic SIV.

Disruption of bbe02 by Insertion of a Luciferase Gene Increases Transformation Efficiency of Borrelia burgdorferi and Allows Live Imaging in Lyme Disease Susceptible C3H Mice.

Lyme disease is the most prevalent tick-borne disease in North America and Europe. The causative agent, Borrelia burgdorferi persists in the white-footed mouse. Infection with B. burgdorferi can cause acute to persistent multisystemic Lyme disease in humans. Some disease manifestations are also exhibited in the mouse model of Lyme disease. Genetic manipulation of B. burgdorferi remains difficult. First, B. burgdorferi contains a large number of endogenous plasmids with unique sequences encoding unknown functions. The presence of these plasmids needs to be confirmed after each genetic manipulation. Second, the restriction modification defense systems, including that encoded by bbe02 gene lead to low transformation efficiency in B. burgdorferi. Therefore, studying the molecular basis of Lyme pathogenesis is a challenge. Furthermore, investigation of the role of a specific B. burgdorferi protein throughout infection requires a large number of mice, making it labor intensive and expensive. To overcome the problems associated with low transformation efficiency and to reduce the number of mice needed for experiments, we disrupted the bbe02 gene of a highly infectious and pathogenic B. burgdorferi strain, N40 D10/E9 through insertion of a firefly luciferase gene. The bbe02 mutant shows higher transformation efficiency and maintains luciferase activity throughout infection as detected by live imaging of mice. Infectivity and pathogenesis of this mutant were comparable to the wild-type N40 strain. This mutant will serve as an ideal parental strain to examine the roles of various B. burgdorferi proteins in Lyme pathogenesis in the mouse model in the future.

Cyclooxygenase 2 activity modulates the severity of murine Lyme arthritis.

Cyclooxygenase (Cox) is a key enzyme in the biosynthetic metabolism of prostaglandins. The inducible isoform of Cox-2 has been implicated in inflammation and its specific inhibition can be used to treat noninfectious inflammatory diseases, such as rheumatoid arthritis. Borrelia burgdorferi, the agent of Lyme disease, can induce joint inflammation. Here we show that B. burgdorferi induced the upregulation of cox-2 gene expression in murine joints at the onset of arthritis in infected mice. The level of mRNA expression correlated with the degree of inflammation. The specific inhibition of Cox-2 diminished the degree of joint inflammation, without affecting B. burgdorferi-specific antibody or cytokine responses. Cox-2 activity is therefore associated with the genesis of infectious arthritis caused by B. burgdorferi.

The mouse as a model for investigation of human granulocytic ehrlichiosis: current knowledge and future directions.

The use of laboratory mice to investigate correlates of infectious disease, including infection kinetics, cellular alterations, cytokine profiles, and immune response in the context of an intact host has expanded exponentially in the last decade. A marked increase in the availability of transgenic mice and research tools developed specifically for the mouse parallels and enhances this research. Human granulocytic ehrlichiosis (HGE) is an emerging, zoonotic disease caused by tick-borne bacteria. The HGE agent (Anaplasma phagocytophila) is one of two recognized pathogens to cause human granulocytic ehrlichiosis (HGE). The mouse model of HGE complements in vitro tissue culture studies, limited in vivo large animal studies, and ex vivo studies of human and ruminant neutrophils, and promises new avenues to approach mechanisms of disease. In the overview reported here, we focus principally on current research into HGE pathogenesis using the mouse model. Included is a discussion of current changes in ehrlichial classification and nomenclature, a review of ehrlichial biology and ecology, and highlights of clinical disease in animals and people.

Use of the P167 recombinant antigen for serodiagnosis of Helicobacter bilis.

Helicobacter bilis is widespread among research mouse colonies. Serodiagnosis of Helicobacter infections involves use of bacterial lysates or membrane antigen preparations that lack specificity, necessitating the need to identify a specific and sensitive antigen. A previously reported recombinant protein (P167) was evaluated for use as an H. bilis-specific antigen for serologic testing. Seventy-six mice naturally infected with Helicobacter spp. were identified from commercially bred or sentinel mice. Infection was confirmed and speciated by use of cecal specimen culture and fecal polymerase chain reaction (PCR) analysis, followed by restriction enzyme digest of the amplicon. Forty-one mice were determined to be monoinfected with H. bilis, 27 mice were determined to be monoinfected with H. hepaticus, and eight mice were infected with another species of Helicobacter. Serum was diluted 1:100 to evaluate the immunoreactivity to enzyme-linked immunosorbent assay preparations of H. bilis membrane extract and the immunodominant C and D fragments of the p167 gene. The sensitivity was greatest for the membrane extract preparation (76%), whereas sensitivity to the P167C and D recombinants was lower (62 and 51%, respectively). However, the specificity of the membrane extract preparation was low (87%), compared with the much improved specificity of the recombinant P167C and D fragments (96 and 96%, respectively). These findings suggest that the recombinant P167C and D fragments of the p167 gene product from H. bilis can be used as specific reagents in the serodiagnosis of H. bilis infection in mice.