A comparison of Bartonella henselae infection in immunocompetent and immunocompromised mice.

Bartonellosis refers to disease caused by the Bartonella genus of bacteria. The breadth of disease manifestations associated with Bartonella is currently expanding and includes regional lymphadenopathy, rheumatic, ocular, and neurological disorders. The dearth of knowledge regarding diagnosis, treatment and pathogenesis of this disease can be partially attributed to the lack of a reliable small animal model for the disease. For this study, Bartonella henselae, the most common species associated with human disease, was injected into Swiss Webster (SW) mice. When the outcome indicated that productive infection did not occur, SCID/Beige (immune compromised) mice were inoculated. While SW mice may potentially harbor an acute infection, less than 10 days in length, the SCID/Beige model provided a sustained infection lasting up to 30-days. These data indicate that SCID/Beige mice can provide a model to study Bartonella infection, therapeutics, and vector dynamics in the future.

Evaluation of the available animal models for Bartonella infections.

The diseases caused by the Bartonella genus of bacteria are clinically diverse, and can be challenging to cure. The study of bartonellosis has been hampered by the lack of a suitable animal model. Preclinical studies for novel therapeutics and a competent host for vector transmission studies are needed to fill critical knowledge gaps. The studies included here are a representation of in vivo Bartonella research and the corresponding challenges. This review examines the current state of available animal models by assessing the success of various model species and strains in Bartonella infection. With a focus on the strengths and weaknesses of current animal models, the importance of these models for improvement of human health and veterinary care is emphasized.

Direct Detection of Borrelia Species in Tissues.

Among the controversies in Lyme disease is the potential for Borrelia spirochetes to persist after guideline-directed antimicrobial therapy. Direct detection of the spirochetes has been essential to explore this phenomenon, given that the infection is often occult and infrequently observed in blood and other body fluids. In addition, the role of spirochetal infection has been examined in the etiology of neurodegenerative diseases through detection in affected tissues. In this chapter, we describe methodology to specifically identify Borrelia DNA, RNA, and intact organism (via protein) in tissue for studies of Lyme Borreliosis.

Targeting Borrelia burgdorferi HtpG with a berserker molecule, a strategy for anti-microbial development.

Conventional antimicrobial discovery relies on targeting essential enzymes in pathogenic organisms, contributing to a paucity of new antibiotics to address resistant strains. Here, by targeting a non-essential enzyme, Borrelia burgdorferi HtpG, to deliver lethal payloads, we expand what can be considered druggable within any pathogen. We synthesized HS-291, an HtpG inhibitor tethered to the photoactive toxin verteporfin. Reactive oxygen species, generated by light, enables HS-291 to sterilize Borrelia cultures by causing oxidation of HtpG, and a discrete subset of proteins in proximity to the chaperone. This caused irreversible nucleoid collapse and membrane blebbing. Tethering verteporfin to the HtpG inhibitor was essential, since free verteporfin was not retained by Borrelia in contrast to HS-291. For this reason, we liken HS-291 to a berserker, wreaking havoc upon the pathogen's biology once selectively absorbed and activated. This strategy expands the druggable pathogenic genome and offsets antibiotic resistance by targeting non-essential proteins.

Superior efficacy of combination antibiotic therapy versus monotherapy in a mouse model of Lyme disease.

Lyme disease (LD) results from the most prevalent tick-borne infection in North America, with over 476,000 estimated cases annually. The disease is caused by Borrelia burgdorferi (Bb) sensu lato which transmits through the bite of Ixodid ticks. Most cases treated soon after infection are resolved by a short course of oral antibiotics. However, 10-20% of patients experience chronic symptoms because of delayed or incomplete treatment, a condition called Post-Treatment Lyme Disease (PTLD). Some Bb persists in PTLD patients after the initial course of antibiotics and an effective treatment to eradicate the persistent Bb is needed. Other organisms that cause persistent infections, such as M. tuberculosis, are cleared using a combination of therapies rather than monotherapy. A group of Food and Drug Administration (FDA)-approved drugs previously shown to be efficacious against Bb in vitro were used in monotherapy or in combination in mice infected with Bb. Different methods of detection were used to assess the efficacy of the treatments in the infected mice including culture, xenodiagnosis, and molecular techniques. None of the monotherapies eradicated persistent Bb. However, 4 dual combinations (doxycycline + ceftriaxone, dapsone + rifampicin, dapsone + clofazimine, doxycycline + cefotaxime) and 3 triple combinations (doxycycline + ceftriaxone+ carbomycin, doxycycline + cefotaxime+ loratadine, dapsone+ rifampicin+ clofazimine) eradicated persistent Bb infections. These results suggest that combination therapy should be investigated in preclinical studies for treating human Lyme disease.

Lyme disease and the pursuit of a clinical cure.

Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most common vector-borne illness in the United States. Many aspects of the disease are still topics of controversy within the scientific and medical communities. One particular point of debate is the etiology behind antibiotic treatment failure of a significant portion (10-30%) of Lyme disease patients. The condition in which patients with Lyme disease continue to experience a variety of symptoms months to years after the recommended antibiotic treatment is most recently referred to in the literature as post treatment Lyme disease syndrome (PTLDS) or just simply post treatment Lyme disease (PTLD). The most commonly proposed mechanisms behind treatment failure include host autoimmune responses, long-term sequelae from the initial Borrelia infection, and persistence of the spirochete. The aims of this review will focus on the in vitro, in vivo, and clinical evidence that either validates or challenges these mechanisms, particularly with regard to the role of the immune response in disease and resolution of the infection. Next generation treatments and research into identifying biomarkers to predict treatment responses and outcomes for Lyme disease patients are also discussed. It is essential that definitions and guidelines for Lyme disease evolve with the research to translate diagnostic and therapeutic advances to patient care.

Repeated Tick Infestations Impair Borrelia burgdorferi Transmission in a Non-Human Primate Model of Tick Feeding.

The blacklegged tick, Ixodes scapularis, is the predominant vector of Borrelia burgdorferi, the agent of Lyme disease in the USA. Natural hosts of I. scapularis such as Peromyscus leucopus are repeatedly infested by these ticks without acquiring tick resistance. However, upon repeated tick infestations, non-natural hosts such as guinea pigs, mount a robust immune response against critical tick salivary antigens and acquire tick resistance able to thwart tick feeding and Borrelia burgdorferi transmission. The salivary targets of acquired tick resistance could serve as vaccine targets to prevent tick feeding and the tick transmission of human pathogens. Currently, there is no animal model able to demonstrate both tick resistance and diverse clinical manifestations of Lyme disease. Non-human primates serve as robust models of human Lyme disease. By evaluating the responses to repeated tick infestation, this animal model could accelerate our ability to define the tick salivary targets of acquired resistance that may serve as vaccines to prevent the tick transmission of human pathogens. Towards this goal, we assessed the development of acquired tick resistance in non-human primates upon repeated tick infestations. We report that following repeated tick infestations, non-human primates do not develop the hallmarks of acquired tick resistance observed in guinea pigs. However, repeated tick infestations elicit immune responses able to impair the tick transmission of B. burgdorferi. A mechanistic understanding of the protective immune responses will provide insights into B. burgdorferi-tick-host interactions and additionally contribute to anti-tick vaccine discovery.

Direct Capture and Early Detection of Lyme Disease Spirochete in Skin with a Microneedle Patch.

Borrelia burgdorferi sensu lato family of spirochetes causes Lyme disease (LD) in animals and humans. As geographic territory of ticks expands across the globe, surveillance measures are needed to measure transmission rates and provide early risk testing of suspected bites. The current standard testing of LD uses an indirect two-step serological assay that detects host immune reactivity. Early detection remains a challenge because the host antibody response develops several weeks after infection. A microneedle (MN) device was developed to sample interstitial fluid (ISF) and capture spirochetes directly from skin. After sampling, the MN patch is easily dissolved in water or TE buffer, and the presence of spirochete DNA is detected by PCR. Performance was tested by spiking porcine ear skin with inactivated Borrelia burgdorferi, which had an approximate recovery of 80% of spirochetes. With further development, this simple direct PCR method could be a transformative approach for early detection of the causative agent of Lyme disease and enable rapid treatment to patients when infection is early, and numbers of systemic spirochetes are low.

Borrelia burgdorferi Migration Assays for Evaluation of Chemoattractants in Tick Saliva.

Uptake of the Lyme disease spirochete by its tick vector requires not only chemical signals present in the tick's saliva but a responsive phenotype by the Borrelia burgdorferi living in the mammalian host. This is the principle behind xenodiagnosis, wherein pathogen is detected by vector acquisition. To study migration of B. burgdorferi toward Ixodes scapularis tick saliva, with the goal of identifying chemoattractant molecules, we tested multiple assays and compared migration of host-adapted spirochetes to those cultured in vitro. We tested mammalian host-adapted spirochetes, along with those grown in culture at 34 °C, for their relative attraction to tick saliva or the nutrient N-acetyl-D-glucosamine (D-GlcNAc) and its dimer chitobiose using two different experimental designs. The host-adapted B. burgdorferi showed greater preference for tick saliva over the nutrients, whereas the cultured incubator-grown B. burgdorferi displayed no significant attraction to saliva versus a significant response to the nutrients. Our results not only describe a validated migration assay for studies of the Lyme disease agent, but provide a further understanding of how growth conditions and phenotype of B. burgdorferi are related to vector acquisition.

Identification of microRNAs in the Lyme Disease Vector Ixodes scapularis.

MicroRNAs (miRNAs) are a class of small non-coding RNAs involved in many biological processes, including the immune pathways that control bacterial, parasitic, and viral infections. Pathogens probably modify host miRNAs to facilitate successful infection, so they might be useful targets for vaccination strategies. There are few data on differentially expressed miRNAs in the black-legged tick Ixodes scapularis after infection with Borrelia burgdorferi, the causative agent of Lyme disease in the United States. Small RNA sequencing and qRT-PCR analysis were used to identify and validate differentially expressed I. scapularis salivary miRNAs. Small RNA-seq yielded 133,465,828 (≥18 nucleotides) and 163,852,135 (≥18 nucleotides) small RNA reads from Borrelia-infected and uninfected salivary glands for downstream analysis using the miRDeep2 algorithm. As such, 254 miRNAs were identified across all datasets, 25 of which were high confidence and 51 low confidence known miRNAs. Further, 23 miRNAs were differentially expressed in uninfected and infected salivary glands: 11 were upregulated and 12 were downregulated upon pathogen infection. Gene ontology and network analysis of target genes of differentially expressed miRNAs predicted roles in metabolic, cellular, development, cellular component biogenesis, and biological regulation processes. Several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including sphingolipid metabolism; valine, leucine and isoleucine degradation; lipid transport and metabolism; exosome biogenesis and secretion; and phosphate-containing compound metabolic processes, were predicted as targets of differentially expressed miRNAs. A qRT-PCR assay was utilized to validate the differential expression of miRNAs. This study provides new insights into the miRNAs expressed in I. scapularis salivary glands and paves the way for their functional manipulation to prevent or treat B. burgdorferi infection.

Characterization of Immunological Responses to Borrelia Immunogenic Protein A (BipA), a Species-Specific Antigen for North American Tick-Borne Relapsing Fever.

Tick-borne relapsing fever (TBRF) is a neglected vector-borne bacterial disease distributed worldwide. Borrelia turicatae, Borrelia parkeri, and Borrelia hermsii are three argasid-borne TBRF species previously implicated in human disease in North America. TBRF is likely underdiagnosed due to its nonspecific symptoms and poorly developed diagnostic tests. Studies suggest that the Borrelia immunogenic protein A (BipA) is specific to TBRF Borrelia but heterogenic between species. In this study, we hypothesized that antibody responses generated to BipA are specific to the North American TBRF species infecting a given animal. To test this, we characterized the expression and localization of native BipA in North American species of TBRF Borrelia. We also infected mice by needle inoculation or tick bite with B. turicatae, B. hermsii, or B. parkeri and evaluated serum sample reactivity to recombinant BipA (rBipA) that was produced from each species. Furthermore, serum samples from nonhuman primates and domestic dogs experimentally infected with B. turicatae were assessed. Lastly, we tested human Lyme disease (LD) serum samples to determine potential cross-reactivity to rBipA generated from B. turicatae, B. parkeri, and B. hermsii. Our findings indicate that rBipA has the potential to distinguish between infections of LD- and TBRF-causing spirochetes and that antibody responses were more robust toward the Borrelia species causing infection. This work further supports that rBipA can likely distinguish between B. turicatae, B. hermsii, and B. parkeri infections in mice, canines, and nonhuman primates. IMPORTANCEBorrelia species transmitted by soft or hard ticks cause tick-borne relapsing fever (TBRF). This is a debilitating disease distributed worldwide but is likely underdiagnosed or misdiagnosed as Lyme disease due to poorly developed diagnostic tests. Borrelia turicatae, Borrelia parkeri, and Borrelia hermsii are three TBRF species previously implicated in human disease in North America. Commonly used diagnostic methods do not identify the species causing infection. In this study, we evaluated the potential of recombinant Borrelia immunogenic protein A (rBipA) as a diagnostic antigen capable of distinguishing between infections of TBRF Borrelia species. We show that serum from mice, canines, and nonhuman primates infected with B. turicatae, B. parkeri, or B. hermsii react more strongly to the rBipA from the species causing infection. Furthermore, sera from Lyme disease patients failed to cross-react with our rBipA proteins, indicating the potential to use rBipA as a species-specific diagnostic antigen for TBRF.

Borreliella burgdorferi Antimicrobial-Tolerant Persistence in Lyme Disease and Posttreatment Lyme Disease Syndromes.

The annual incidence of Lyme disease, caused by tick-transmitted Borreliella burgdorferi, is estimated to be at least 476,000 cases in the United States and many more worldwide. Ten to 20% of antimicrobial-treated Lyme disease patients display posttreatment Lyme disease syndrome (PTLDS), a clinical complication whose etiology and pathogenesis remain uncertain. Autoimmunity, cross-reactivity, molecular mimicry, coinfections, and borrelial tolerance to antimicrobials/persistence have been hypothesized and studied as potential causes of PTLDS. Studies of borrelial tolerance/persistence in vitro in response to antimicrobials and experimental studies in mice and nonhuman primates, taken together with clinical reports, have revealed that B. burgdorferi becomes tolerant to antimicrobials and may sometimes persist in animals and humans after the currently recommended antimicrobial treatment. Moreover, B. burgdorferi is pleomorphic and can generate viable-but-nonculturable bacteria, states also involved in antimicrobial tolerance. The multiple regulatory pathways and structural genes involved in mediating this tolerance to antimicrobials and environmental stressors by persistence might include the stringent (rel and dksA) and host adaptation (rpoS) responses, sugar metabolism (glpD), and polypeptide transporters (opp). Application of this recently reported knowledge to clinical studies can be expected to clarify the potential role of bacterial antibacterial tolerance/persistence in Lyme disease and PTLDS.

An Exploratory Study on the Microbiome of Northern and Southern Populations of Ixodes scapularis Ticks Predicts Changes and Unique Bacterial Interactions.

The black-legged tick (Ixodes scapularis) is the primary vector of Borrelia burgdorferi, the causative agent of Lyme disease in North America. However, the prevalence of Lyme borreliosis is clustered around the Northern States of the United States of America. This study utilized a metagenomic sequencing approach to compare the microbial communities residing within Ix. scapularis populations from northern and southern geographic locations in the USA. Using a SparCC network construction model, we performed potential interactions between members of the microbial communities from Borrelia burgdorferi-infected tissues of unfed and blood-fed ticks. A significant difference in bacterial composition and diversity was found between northern and southern tick populations. The network analysis predicted a potential antagonistic interaction between endosymbiont Rickettsia buchneri and Borrelia burgdorferi sensu lato. The network analysis, as expected, predicted significant positive and negative microbial interactions in ticks from these geographic regions, with the genus Rickettsia, Francisella, and Borreliella playing an essential role in the identified clusters. Interactions between Rickettsia buchneri and Borrelia burgdorferi sensu lato need more validation and understanding. Understanding the interplay between the microbiome and tick-borne pathogens within tick vectors may pave the way for new strategies to prevent tick-borne infections.

Recent Progress in Lyme Disease and Remaining Challenges.

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.

Improving rigor and reproducibility in nonhuman primate research.

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.

Antibiotic Susceptibility of Bartonella Grown in Different Culture Conditions.

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.

Report of the Pathogenesis and Pathophysiology of Lyme Disease Subcommittee of the HHS Tick Borne Disease Working Group.

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.

Detecting Borrelia Spirochetes: A Case Study With Validation Among Autopsy Specimens.

The complex etiology of neurodegenerative disease has prompted studies on multiple mechanisms including genetic predisposition, brain biochemistry, immunological responses, and microbial insult. In particular, Lyme disease is often associated with neurocognitive impairment with variable manifestations between patients. We sought to develop methods to reliably detect Borrelia burgdorferi, the spirochete bacteria responsible for Lyme disease, in autopsy specimens of patients with a history of neurocognitive disease. In this report, we describe the use of multiple molecular detection techniques for this pathogen and its application to a case study of a Lyme disease patient. The patient had a history of Lyme disease, was treated with antibiotics, and years later developed chronic symptoms including dementia. The patient's pathology and clinical case description was consistent with Lewy body dementia. B. burgdorferi was identified by PCR in several CNS tissues and by immunofluorescent staining in the spinal cord. These studies offer proof of the principle that persistent infection with the Lyme disease spirochete may have lingering consequences on the CNS.

Blocking Borrelia burgdorferi transmission from infected ticks to nonhuman primates with a human monoclonal antibody.

Disrupting transmission of Borrelia burgdorferi sensu lato complex (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 mAb, 2217, prevents transmission of B. burgdorferi from infected ticks in animal models. Maintenance of a protective plasma concentration of a human mAb for tick season presents a significant challenge for a preexposure prophylaxis strategy. Here, we describe the optimization of mAb 2217 by amino acid substitutions (2217LS: M428L and N434S) in the Fc domain. The LS mutation led to a 2-fold 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 revealed that 2217 bound an epitope that was 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 preexposure 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.