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Nonhuman primates (NHPs) are a critical component of translational/preclinical biomedical research due to the strong similarities between NHP and human physiology and disease pathology. In some cases, NHPs represent the most appropriate, or even the only, animal model for complex metabolic, neurological, and infectious diseases. The increased demand for and limited availability of these valuable research subjects requires that rigor and reproducibility be a prime consideration to ensure the maximal utility of this scarce resource. Here, we discuss a number of approaches that collectively can contribute to enhanced rigor and reproducibility in NHP research.

Lyme disease (also known as Lyme borreliosis) is the most common vector-borne disease in the United States with an estimated 476,000 cases per year. While historically, the long-term impact of Lyme disease on patients has been controversial, mounting evidence supports the idea that a substantial number of patients experience persistent symptoms following treatment. The research community has largely lacked the necessary funding to properly advance the scientific and clinical understanding of the disease, or to develop and evaluate innovative approaches for prevention, diagnosis, and treatment. Given the many outstanding questions raised into the diagnosis, clinical presentation and treatment of Lyme disease, and the underlying molecular mechanisms that trigger persistent disease, there is an urgent need for more support. This review article summarizes progress over the past 5 years in our understanding of Lyme and tick-borne diseases in the United States and highlights remaining challenges.

In vivo diagnostic imaging of bacterial infections is currently reliant on targeting their metabolic pathways, an ineffective method to identify microbial species with low metabolic activity. Here, we establish HS-198 as a small-molecule fluorescent conjugate that selectively targets the highly conserved bacterial protein HtpG (high-temperature protein G), within Borrelia burgdorferi, the bacterium responsible for Lyme disease. We describe the use of HS-198 to target morphologic forms of B. burgdorferi in both the logarithmic growth phase and the metabolically dormant stationary phase as well as in inactivated spirochetes. Furthermore, in a murine infection model, systemically injected HS-198 identified B. burgdorferi as revealed by imaging in postnecropsy tissue sections. These findings demonstrate how small-molecule probes directed at conserved bacterial protein targets can function to identify the microbe using noninvasive imaging and potentially as scaffolds to deliver antimicrobial agents to the pathogen.

An understanding of the pathogenesis and pathophysiology of Lyme disease is key to the ultimate care of patients with Lyme disease. To better understand the various mechanisms underlying the infection caused by Borrelia burgdorferi, the Pathogenesis and Pathophysiology of Lyme Disease Subcommittee was formed to review what is currently known about the pathogenesis and pathophysiology of Lyme disease, from its inception, but also especially about its ability to persist in the host. To that end, the authors of this report were assembled to update our knowledge about the infectious process, identify the gaps that exist in our understanding of the process, and provide recommendations as to how to best approach solutions that could lead to a better means to manage patients with persistent Lyme disease.

Disrupting transmission of Borrelia burgdorferi (B. burgdorferi ) from infected ticks to humans is one strategy to prevent the significant morbidity from Lyme disease. We have previously shown that an anti-OspA human monoclonal antibody, 2217, prevents transmission of B. burgdorferi from infected ticks in animal models. Maintenance of a protective plasma concentration of a human monoclonal antibody for tick season presents a significant challenge for a pre-exposure prophylaxis strategy. Here, we describe the optimization of 2217 by amino acid substitutions (LS, M428L and N434S) into the Fc domain. The LS mutation led to a twofold-increase in half-life in cynomolgus monkeys. In a rhesus macaque model, 2217LS protected animals from tick transmission of spirochetes at a dose of 3 mg/kg. Crystallographic analysis of Fab in complex with OspA reveals that 2217 binds a novel epitope that is highly conserved among the B. burgdorferi, B. garinii, and B. afzelii species. Unlike most vaccines that may require boosters to achieve protection, our work supports the development of 2217LS as an effective pre-exposure prophylaxis in Lyme-endemic regions with a single dose at the beginning of tick season offering immediate protection that remains for the duration of exposure risk. .

Bartonellosis is caused by a Gram-negative intracellular bacterium with a zoonotic transmission. The disease, caused by any of several genospecies of Bartonella can range from a benign, self-limited condition to a highly morbid and life-threatening illness. The current standard of care antibiotics are generally effective in acute infection, these include azithromycin or erythromycin, doxycycline, gentamicin, rifampin, and ciprofloxacin. However, treatment of chronic infection remains problematic. We tested six different antibiotics for their ability to stop the growth of Bartonella sp. in the standard insect media and in an enrichment media. All antibiotics (ceftriaxone, doxycycline, gentamycin, azithromycin, ampicillin, and azlocillin) had minimum inhibitory concentrations (MICs) below 0.5 µg/mL in the BAPGM enrichment media but were ineffective at inhibiting growth when the standard insect media was used. Azlocillin was the most potent, with a MIC of 0.01 µg/mL. When Bartonella was tested under intracellular growth conditions, none of the antibiotics were efficacious singly. However, growth inhibition was observed when azlocillin and azithromycin were combined. These studies illustrate the impact of growth medium and intracellular environment on antibiotic susceptibility testing and indicate that azlocillin combined with azithromycin may be an effective drug combination for the treatment of Bartonellosis.

In vivo diagnostic imaging of bacterial infections is currently reliant on targeting their metabolic pathways, an ineffective method to identify microbial species with low metabolic activity. Here we establish HS-198 as a small molecule-fluorescent conjugate that selectively targets the highly conserved bacterial protein, HtpG (High temperature protein G), within B. burgdorferi, the bacteria responsible for Lyme Disease. We describe the use of HS-198 to target morphologic forms of B. burgdorferi in both the logarithmic growth phase and the metabolically dormant stationary phase. Furthermore, in a murine infection model, systemically injected HS-198 identified B. burgdorferi as revealed by imaging in post necropsy tissue sections. These findings demonstrate how small molecule probes directed at conserved bacterial protein targets can function to identify the microbe using non-invasive imaging and potentially as scaffolds to deliver antimicrobial agents to the pathogen.

Aims To investigate the immunoglobulin (Ig) G response after being fed upon by Cimex lectularius L. Methods and results Participants were fed upon by three male C lectularius insects weekly for a month. Blood was obtained before the feeding and at the last feeding, which was used for immunoblots against bed bug salivary gland extract, with antihuman Immunoglobulin G (IgG) secondary antibodies. No consistent IgG changes developed in 11 humans serially fed upon by C lectularius. Two participants had new IgG responses to proteins at molecular weights of approximately 12-13 kDa, and one had an IgG response to a protein at approximately 40 kDa. At the last study visit, more intense IgG bands to proteins at molecular weights of 12-13 kDa had developed in 55% of participants (6/11) and at molecular weights of ≈30, ≈40 and ≈70 kDa in 45% (5/11) compared with the first study visit. Nitrophorin and apyrase were the most common C lectularius proteins identified with liquid chromatography-tandem mass spectrometry in both crushed bed bug salivary gland extract and post-bed bug feeding extract. Conclusions Human participants did not have consistent IgG responses to crushed C lectularius salivary gland extract.

Objectives Borrelia miyamotoi is a relapsing fever Borrelia, transmitted by hard (Ixodes) ticks, which are also the main vector for Borrelia burgdorferi. A widely used test for serodiagnosis of Lyme borreliosis is an enzyme immunoassay (EIA) based on the C6 peptide of the B. burgdorferi sl VlsE protein. We set out to study C6 reactivity upon infection with B. miyamotoi in a large well-characterized set of B. miyamotoi disease (BMD) patient sera and in experimental murine infection. Methods We performed in silico analyses, comparing the C6-peptide to immunodominant B. miyamotoi variable large proteins (Vlps). Next, we determined C6 reactivity in sera from mice infected with B. miyamotoi and in a unique longitudinal set of 191 sera from 46 BMD patients. Results In silico analyses revealed similarity of the C6 peptide to domains within B. miyamotoi Vlps. Cross-reactivity against the C6 peptide was confirmed in 21 out of 24 mice experimentally infected with B. miyamotoi. Moreover, 35 out of 46 BMD patients had a C6 EIA Lyme index higher than 1.1 (positive). Interestingly, 27 out of 37 patients with a C6 EIA Lyme index higher than 0.9 (equivocal) were negative when tested for specific B. burgdorferi sl antibodies using a commercially available immunoblot. Conclusions We show that infection with B. miyamotoi leads to cross-reactive antibodies to the C6 peptide. Since BMD and Lyme borreliosis are found in the same geographical locations, caution should be used when relying solely on C6 reactivity testing. We propose that a positive C6 EIA with negative immunoblot, especially in patients with fever several weeks after a tick bite, warrants further testing for B. miyamotoi.

Nonhuman primates currently serve as the best experimental model for Lyme disease because of their close genetic homology with humans and demonstration of all three phases of disease after infection with Borrelia burgdorferi. We investigated the pathology associated with late disseminated Lyme disease (12 to 13 months after tick inoculation) in doxycycline-treated (28 days; 5 mg/kg, oral, twice daily) and untreated rhesus macaques. Minimal to moderate lymphoplasmacytic inflammation, with a predilection for perivascular spaces and collagenous tissues, was observed in multiple tissues, including the cerebral leptomeninges, brainstem, peripheral nerves from both fore and hind limbs, stifle synovium and perisynovial adipose tissue, urinary bladder, skeletal muscle, myocardium, and visceral pericardium. Indirect immunofluorescence assays that combined monoclonal (outer surface protein A) and polyclonal antibodies were performed on all tissue sections that contained inflammation. Rare morphologically intact spirochetes were observed in the brains of two treated rhesus macaques, the heart of one treated rhesus macaque, and adjacent to a peripheral nerve of an untreated animal. Borrelia antigen staining of probable spirochete cross sections was also observed in heart, skeletal muscle, and near peripheral nerves of treated and untreated animals. These findings support the notion that chronic Lyme disease symptoms can be attributable to residual inflammation in and around tissues that harbor a low burden of persistent host-adapted spirochetes and/or residual antigen.

Recent studies have shown that Borrelia burgdorferi can form antibiotic-tolerant persisters in the presence of microbiostatic drugs such as doxycycline. Precisely how this occurs is yet unknown. Our goal was to examine gene transcription by B. burgdorferi following doxycycline treatment in an effort to identify both persister-associated genes and possible targets for antimicrobial intervention. To do so, we performed next-generation RNA sequencing on doxycycline-treated spirochetes and treated spirochetes following regrowth, comparing them to untreated B. burgdorferi. A number of genes were perturbed and most of those which were statistically significant were down-regulated in the treated versus the untreated or treated/re-grown. Genes upregulated in the treated B. burgdorferi included a number of Erp genes and rplU, a 50S ribosomal protein. Among those genes associated with post-treatment regrowth were bba74 (Oms28), bba03, several peptide ABC transporters, ospA, ospB, ospC, dbpA and bba62. Studies are underway to determine if these same genes are perturbed in B. burgdorferi treated with doxycycline in a host environment.

As Ixodes ticks spread to new regions, the incidence of Lyme disease (LD) in companion animals and humans will increase. Preventive strategies for LD in canines center on vaccination and tick control (acaricides). Both subunit and bacterin based LD veterinary vaccines are available. Outer surface protein C (OspC), a potent immunogen and dominant early antigen, has been demonstrated to elicit protective antibody (Ab) responses. However, a single OspC protein elicits a relatively narrow range of protection. There are conflicting reports as to whether the immunodominant epitopes of OspC reside within variable or conserved domains. A detailed understanding of the antigenic determinants of OspC is essential for understanding immune responses to this essential virulence factor and vaccinogen. Here, we investigate the contribution of the conserved C-terminal C10 motif in OspC triggered Ab responses. Using a panel of diverse recombinant full length OspC proteins and their corresponding C10 deletion variants (OspCΔC10), we demonstrate that the C10 motif does not significantly contribute to immunization or infection induced Ab responses in rabbits, rats, canines, horses and non-human primates. Furthermore, the C10 motif is not required to trigger potent bactericidal Ab responses. This study provides insight into the antigenic structure of OspC. The results enhance our understanding of immune responses that develop during infection or upon vaccination and have implications for interpretation of LD diagnostic assays that employ OspC.

The global public health impact of relapsing fever (RF) spirochetosis is significant, since the pathogens exist on five of seven continents. The hallmark sign of infection is episodic fever and the greatest threat is to the unborn. With the goal of better understanding the specificity of B-cell responses and the role of immune responses in pathogenicity, we infected rhesus macaques with Borrelia turicatae (a new world RF spirochete species) by tick bite and monitored the immune responses generated in response to the pathogen. Specifically, we evaluated inflammatory mediator induction by the pathogen, host antibody responses to specific antigens, and peripheral lymphocyte population dynamics. Our results indicate that B. turicatae elicits from peripheral blood cells key inflammatory response mediators (interleukin-1β and tumor necrosis factor alpha), which are associated with preterm abortion. Moreover, a global decline in peripheral B-cell populations was observed in all animals at 14 days postinfection. Serological responses were also evaluated to assess the antigenicity of three surface proteins: BipA, BrpA, and Bta112. Interestingly, a distinction was observed between antibodies generated in nonhuman primates and mice. Our results provide support for the nonhuman primate model not only in studies of prenatal pathogenesis but also for diagnostic and vaccine antigen identification and testing.

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Lyme disease (Borrelia burgdorferi infection) is increasingly recognized as a significant source of morbidity worldwide. Here, we show that blood plasmablasts and CD27− memory B cells are elevated in untreated Lyme disease, with higher plasmablast levels associated with more rapid resolution of clinical symptoms. Stronger serum reactivity to surface proteins and peptides from B. burgdorferi was also associated with faster resolution of clinical symptoms. Through molecular identifier-enabled antibody heavy-chain sequencing of bulk B cells and single-cell paired-chain antibody sequencing of blood plasmablasts, we characterized immunoglobulin gene usage patterns specific to B. burgdorferi infection. Recombinantly expressed antibodies from expanded lineages bound B. burgdorferi antigens, confirming that these clones are driven by the infection. Furthermore, recombinant sequence-derived antibodies were functional, inhibiting growth of B. burgdorferi in vitro. Elevations and clonal expansion of blood plasmablasts were associated with rapid return to health, while poor plasmablast responses were associated with a longer duration of symptoms following treatment. Plasmablasts induced by B. burgdorferi infection showed preferential antibody gene segment usage, while bulk sequencing of total B cells revealed convergent CDR3 motifs specific to B. burgdorferi-infected patients. Our results show that robust plasmablast responses encoding Bb-static antibodies are associated with more rapid resolution of Lyme disease, and these antibodies could provide the basis for next-generation therapeutics for Lyme disease.

Background Moraxella macacae is a recently described bacterial pathogen that causes epistaxis or so-called bloody nose syndrome in captive macaques. The aim of this study was to develop specific molecular diagnostic assays for M. macacae and to determine their performance characteristics. Methods We developed six real-time PCR assays on the Roche LightCycler. The accuracy, precision, selectivity, and limit of detection (LOD) were determined for each assay, in addition to further validation by testing nasal swabs from macaques presenting with epistaxis at the Tulane National Primate Research Center. Results All assays exhibited 100% specificity and were highly sensitive with an LOD of 10 fg for chromosomal assays and 1 fg for the plasmid assay. Testing of nasal swabs from 10 symptomatic macaques confirmed the presence of M. macacae in these animals. Conclusions We developed several accurate, sensitive, and species-specific real-time PCR assays for the detection of M. macacae in captive macaques.

The hallmark of disease caused by tick- and louse-borne relapsing fever due to Borrelia infection is cyclic febrile episodes, which in humans results in severe malaise and may lead to death. To evaluate the pathogenesis of relapsing fever due to spirochetes in an animal model closely related to humans, disease caused by Borrelia turicatae after tick bite was compared in 2 rhesus macaques in which radiotelemetry devices that recorded body temperatures in 24-hour increments were implanted. The radiotelemetry devices enabled real-time acquisition of core body temperatures and changes in heart rates and electrocardiogram intervals for 28 consecutive days without the need to constantly manipulate the animals. Blood specimens were also collected from all animals for 14 days after tick bite, and spirochete densities were assessed by quantitative polymerase chain reaction. The complexity of disease caused by relapsing-fever spirochetes was demonstrated in the nonhuman primates monitored in real time. The animals experienced prolonged episodes of hyperthermia and hypothermia; disruptions in their diurnal patterns and repolarization of the heart were also observed. This is the first report of the characterizing disease progression with continuous monitoring in an animal model of relapsing fever due to Borrelia infection.

The systematically difficult task of diagnosing Lyme disease can be simplified by sensitive and specific laboratory tests. The currently recommended two-tier test for serology is highly specific but falls short in sensitivity, especially in the early acute phase. We previously examined serially collected serum samples from Borrelia burgdorferi -infected rhesus macaques and defined a combination of antigens that could be utilized for detection of infection at all phases of disease in humans. The five B. burgdorferi antigens, consisting of OspC, OspA, DbpA, OppA2, and the C6 peptide, were combined into a fluorescent cytometric bead-based assay for the detection of B. burgdorferi antigen-specific IgG antibodies. Samples from Lyme disease patients and controls were used to determine the diagnostic value of this assay. Using this sample set, we found that our five-antigen multiplex IgG assay exhibited higher sensitivity (79.5%) than the enzyme immunoassay (EIA) (76.1%), the two-tier test (61.4%), and the C6 peptide enzyme-linked immunosorbent assay (ELISA) (77.2%) while maintaining specificity over 90%. When detection of IgM was added to the bead-based assay, the sensitivity improved to 91%, but at a cost of reduced specificity (78%). These results indicate that the rational combination of antigens in our multiplex assay may offer an improved serodiagnostic test for Lyme disease.

Background The prolonged feeding process of ixodid ticks, in combination with bacterial transmission, should lead to a robust inflammatory response at the blood-feeding site. Yet, factors present in tick saliva may down-regulate such responses, which may be beneficial to spirochete transmission. The primary goal of this study was to test the hypothesis that tick saliva, in the context of Borrelia burgdorferi, can have widespread effects on the production of immune mediators in skin. Methods A cross-section of tick feeding on skin was examined histologically. Human THP-1 cells stimulated with B. burgdorferi and grown in the presence or absence of tick saliva were examined by human DNA microarray, cytokine bead array, sandwich ELISA, and qRT-PCR. Similar experiments were also conducted using dermal fibroblasts. Results Tick feeding on skin showed dermal infiltration of histiocytes and granulocytes at the bite location. Changes in monocytic transcript levels during co-culture with B. burgdorferi and saliva indicated that tick saliva had a suppressive effect on the expression of certain pro-inflammatory mediators, such as IL-8 (CXCL8) and TLR2, but had a stimulatory effect on specific molecules such as the Interleukin 10 receptor, alpha subunit (IL-10RA), a known mediator of the immunosuppressive signal of IL-10. Stimulated cell culture supernatants were analyzed via antigen-capture ELISA and cytokine bead array for inflammatory mediator production. Treatment of monocytes with saliva significantly reduced the expression of several key mediators including IL-6, IL-8 and TNF-alpha. Tick saliva had an opposite effect on dermal fibroblasts. Rather than inhibiting, saliva enhanced production of pro-inflammatory mediators, including IL-8 and IL-6 from these sentinel skin cells. Conclusions The effects of ixodid tick saliva on resident skin cells is cell type-dependent. The response to both tick and pathogen at the site of feeding favors pathogen transmission, but may not be wholly suppressed by tick saliva. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1638-7) contains supplementary material, which is available to authorized users.

Background Following administration of an antibiotic, the concentration in blood changes over time and is dependent on the type of antibiotic, the route and species of the individual. The most relevant pharmacodynamic property of a bacteriostatic antibiotic such as doxycycline is the minimum inhibitory concentration (MIC), whereas pharmacokinetics may include rates of absorption and elimination from blood. Methods We determined serum concentrations of doxycycline following administration of 5 mg/kg in two macaques. Results The area under the concentration–time curve over 24 hours (AUC0–24) following two doses was extrapolated from the curve over 12 hours following a single dose, with the purpose of calculating the AUC0–24:MIC. Conclusions Other than a somewhat faster rate of elimination, the PK-PD values for doxycycline in macaques appears similar to those determined for humans. This information will be valuable for treating disease in macaques and for research in bacterial infection models that use macaques.

Bacteria of the genus Moraxella have been isolated from a variety of mammalian hosts. In a prior survey of bacteria that colonize the rhesus macaque nasopharynx, performed at the Tulane National Primate Research Center, organisms of the Moraxella genus were isolated from animals with epistaxis, or “bloody nose syndrome.” They were biochemically identified as Moraxella catarrhalis, and cryopreserved. Another isolate was obtained from an epistatic cynomolgus macaque at the U.S. Army Medical Research Institute of Infectious Diseases. Based on differences in colony and cell morphologies between rhesus and human M. catarrhalis isolates, we hypothesized that the nonhuman primate Moraxella might instead be a different species. Despite morphological differences, the rhesus isolates, by several biochemical tests, were indistinguishable from M. catarrhalis. Analysis of the cynomolgus isolate by Vitek 2 Compact indicated that it belonged to a Moraxella group, but could not differentiate among species. However, sequencing of the 16S ribosomal RNA gene from four representative rhesus isolates and the cynomolgus isolate showed closest homology to Moraxella lincolnii, a human respiratory tract inhabitant, with 90.16% identity. To examine rhesus macaques as potential hosts for M. catarrhalis, eight animals were inoculated with human M. catarrhalis isolates. Only one of the animals was colonized and showed disease, whereas four of four macaques became epistatic after inoculation with the rhesus Moraxella isolate. The nasopharyngeal isolates in this study appear uniquely adapted to a macaque host and, though they share many of the phenotypic characteristics of M. catarrhalis, appear to form a genotypically distinct species.

Detection of antibody to C6, a 26–amino acid peptide that reproduces the sequence of the sixth invariable region (IR6) within the central domain of the VlsE protein of Borrelia burgdorferi sensu lato, is currently used for the serologic diagnosis of Lyme disease [1–4]. A potential concern with a test based on a single peptide is the possibility of sequence variation and a consequent lack of antigenic cross-reactivity among pathogenic strains of B. burgdorferi sensu lato, leading to reduced diagnostic sensitivity in some infected patients. B. burgdorferi sensu stricto (hereafter referred to as B. burgdorferi) isolates can be categorized into 3 distinct ribosomal spacer restriction fragment length polymorphism genotypes (RSTs) on the basis of analysis of the 16S-23S ribosomal DNA (rDNA) spacer region [5, 6]. North American isolates of B. burgdorferi can also be differentiated into at least 16 major genetic groups on the basis of variability in the genetic sequence encoding outer surface protein C (OspC) [7, 8]. Studies employing these methods have demonstrated that pathogenicity is dependent at least in part on the genotype of B. burgdorferi causing the infection; for example, evidence indicates that RST1 and RST2 are more likely to be associated with hematogenous dissemination in humans than is RST3 [6]. Although serologic testing of patients with erythema migrans is not routinely recommended for the management of these patients, serum samples from such patients are often used to validate or compare serologic assays, because this is the most common manifestation of Lyme disease [9], and it is the only manifestation for which the diagnosis can frequently be confirmed by the microbiologic gold standard of recovering B. burgdorferi on culture [10]. In a previous study [11], we demonstrated the comparable sensitivity of a C6 assay in 79 patients from North America with erythema migrans, irrespective of the RST genotype that caused the infection. The current study expands on the previous study by increasing the number of patient serum samples evaluated and by also considering the ospC genotype of the infecting strain of B. burgdorferi. In addition, the effect of the genotype of B. burgdorferi on the sensitivity of 2-tier testing was evaluated.

Infectivity and persistence by Borrelia burgdorferi , the etiologic agent of Lyme disease, rely stringently on regulatory events. Among these is the downregulation of lipoprotein antigen expression, exemplified by outer surface protein C (OspC), at the advent of specific immunity in the mammalian host. B. burgdorferi spirochetes that lack the linear plasmid 28-1 (lp28-1) succumb to the host's immune response. We thus explored the notion that these two phenomena were related—that lp28-1 − organisms fail to downregulate ospC and thus are cleared following the appearance of anti-OspC antibody in the murine host. The lp-28-1 − isolate and a wild-type (wt) isolate bearing the complete set of plasmids were grown in dialysis membrane chambers that were implanted into rat peritoneal cavities. Analysis of mRNA and protein from these cultures showed that OspC expression levels by lp28-1 − organisms are abnormally high in vivo. A time course analysis of ospC expression in tissues following infection indicates also that temporal diminution of the dominant antigen OspC is impaired in lp28-1 − spirochetes. Finally, passive transfer of monoclonal OspC-specific antibody into SCID mice 8 days postinfection cleared lp28-1 − spirochetes, yet the wt organisms persisted in a majority of animals. These findings indicate that incomplete repression of OspC by lp28-1 − organisms renders them susceptible to immune-mediated clearance. The lp28-1 plasmid must harbor one or more genes involved in OspC downregulation.

Borrelia burgdorferi, the Lyme disease pathogen, employs several immune-evasive strategies to survive in mammals. Unlike mice, major reservoir hosts for B. burgdorferi, rabbits are considered to be nonpermissive hosts for persistent infection. Antigenic variation of the VlsE molecule is a probable evasion strategy known to function in mice. The invariable region 6 (IR6) and carboxyl-terminal domain (Ct) of VlsE elicit dominant antibody responses that are not protective, perhaps to function as decoy epitopes that protect the spirochete. We sought to determine if either of these characteristics of VlsE differed in rabbit infection, contributing to its reputed nonpermissiveness. VlsE recombination was observed in rabbits that were given inoculations with either cultured or host-adapted spirochetes. Early observations showed a lack of anti-C6 (a peptide encompassing the IR6 region) response in most rabbits, so the anti-Ct and anti-C6 responses were monitored for 98 weeks. Anti-C6 antibody appeared as late as 20 weeks postinoculation, and the anti-Ct response, evident within the first 2 weeks, oscillated for prolonged periods of time. These observations, together with the recovery of cultivable spirochetes from tissue of one animal at 98 weeks postinoculation, challenge the notion that the rabbit cannot harbour a long-term B. burgdorferi infection.

Lyme borreliosis (LB) is a disease for which antibody-based detection assays are often required for diagnosis. The variable surface molecule VlsE and IR6, one of its invariable regions, are commonly targeted by the antibody response in infected individuals. A series of enzyme-linked immunosorbent assays was performed to comparatively examine the antibody responses of North American LB patients (n= 37) to VlsE and invariable segments of this molecule. Both immunoglobulin M (IgM) and IgG responses to full-length VlsE and to peptides reproducing invariable regions 2, 4, and 6, as well as the invariable domains at the amino and carboxyl termini of VlsE, were assessed. The proportions and specificities of reactivity to the invariable segments were tested by using cognate peptides as competitors for VlsE binding by patient serum antibodies. IR6 epitopes (by the C6 peptide) were found to dominate the response to invariable segments. IR6 (C6)-specific antibodies were detected in 78% of the serum specimens, whereas n= 8) that contained IgG antibodies reacting with both C6 and VlsE was assessed in competition experiments, using C6 as a competitor. Only half of these specimens contained IgG antibodies whose binding to VlsE could be inhibited >50% by competition with the added C6 peptide. The median percent inhibition was 45.5%. These findings indicate that IR6 epitopes are largely concealed from the VlsE molecular surface and that full-length VlsE-based diagnosis likely detects antibodies to conformational and/or variable region epitopes.

Detection of antibody to C6, a peptide that reproduces the sequence of the sixth invariable region within the central domain of the VlsE protein of Borrelia burgdorferi, is used currently for the serologic diagnosis of Lyme disease in humans. B. burgdorferi isolates taken from infected humans can be categorized into specific genetic subtypes (designated RST1, -2, and -3) by restriction fragment length polymorphisms in the 16S to 23S rRNA spacer sequence. Many of these, usually categorized as RST2, retain only segments of the linear plasmid lp28-1, which encodes VlsE. The VlsE genetic region is retained, but altered expression of this molecule could affect diagnosis by the C6 enzyme-linked immunosorbent assay (ELISA). Serum samples from patients infected with each of the three genotypes and from mice infected with three RST2 isolates were tested with the C6 ELISA. Such isolates elicited marked C6 responses in infected mice. The sensitivity of C6 antibody detection in patients infected with RST2 spirochetes was statistically indistinguishable from detection of RST1 and RST3 infections. These findings demonstrate that diagnosis by C6 ELISA remains effective for infection with all B. burgdorferi genotypes, including those with incomplete lp28-1 plasmids. The clinical progression of Lyme disease, a tick-borne illness that is caused by the spirochete Borrelia burgdorferi, is divided into early localized, early disseminated, and late stages. During the early localized phase, the disease usually manifests by a characteristic skin lesion, erythema migrans (EM). Several days or weeks later, the spirochetes may spread hematogenously and patients may develop early disseminated disease with dermatologic, cardiac, neurologic, and/or rheumatologic involvement. During the early disseminated phase, some patients exhibit dermatologic signs that appear as multiple EM (24, 27, 29, 33). Late disease presents mainly with arthritis or neurologic manifestations (28). Evidence has been put forward to suggest that a major determinant of the risk of B. burgdorferi dissemination in Lyme disease patients with EM is the genotype of the infecting spirochetal strain. Three genotypes have been identified on the basis of restriction fragment length polymorphisms in the rRNA spacer region and have been designated RST1, -2, and -3 (16). Patients infected with RST1 spirochetes had the highest proportion of blood culture positivity (43%), those infected with RST2 spirochetes were intermediate (20%), and those with RST3 had the lowest proportion (3%). In general, persons with an RST1 infection had more symptoms and symptoms of greater severity than those with either RST2 or RST3 infection (32). Mice that were inoculated with RST1 organisms were significantly more likely to yield cultivable spirochetes from different organs and showed a significantly higher prevalence of both carditis and arthritis than did animals inoculated with RST3 spirochetes (30); RST2 isolates were not assessed. When the plasmid contents of the different human infectious types were assessed, RST2 spirochetes emerged as