scholarly journals Microfluidic Assays for Probing Neutrophil-Borrelia Interactions in Blood During Lyme Disease

2021 ◽  
pp. 1-11
Author(s):  
Sinan Muldur ◽  
Felix Ellett ◽  
Anika L. Marand ◽  
Charles Marvil ◽  
John A. Branda ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Human Neutrophils ◽  
Complement Factor ◽  
Complement Receptors ◽  
Blood Samples ◽  
Spontaneous Motility ◽  
Healthy Donors ◽  
Highly Sensitive ◽  
Microfluidic Assays

Human neutrophils are highly sensitive to the presence of Borrelia burgdorferi (Bb), the agent of Lyme disease (LD), in tissues. Although Bb is also found in the blood of LD patients, far less is known about how neutrophils respond to Bb in the presence of blood. In this study, we employed microfluidic tools to probe the interaction between human neutrophils and Bb and measured the activation of human neutrophils in blood samples from patients. We found that neutrophils migrate vigorously toward Bb in the presence of serum, and this process was complement-dependent. Preventing complement factor 5 cleavage or blocking complement receptors decreased neutrophil’s ability to interact with Bb. We also found that spiking Bb directly into the blood from healthy donors induced spontaneous neutrophil motility. This response to Bb was also complement-dependent. Preventing complement factor 5 cleavage decreased spontaneous neutrophil motility in Bb-spiked blood. Moreover, we found that neutrophils in blood samples from acute LD patients displayed spontaneous motility patterns similar to those observed in Bb-spiked samples. Neutrophil motility was more robust in blood samples from LD patients than that measured in healthy and ill controls, validating the utility of the microfluidic assay for the study of neutrophil-Bb interactions in the presence of blood.

scholarly journals Tube Phagocytosis, a Novel Way for Neutrophils To Phagocytize Borrelia burgdorferi

1998 ◽  
Vol 66 (7) ◽  
pp. 3433-3435 ◽  
Author(s):  
Juha Suhonen ◽  
Kaija Hartiala ◽  
Matti K. Viljanen
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Dark Field ◽  
Human Neutrophils ◽  
Video Technology ◽  
Dark Field Microscopy

ABSTRACT Interactions between human neutrophils and Borrelia burgdorferi, the Lyme disease spirochete, were studied by dark-field microscopy combined with video technology. A previously unrecognized mechanism for neutrophils to phagocytize the spirochete was discovered. During phagocytosis, the spirochete attaches to the neutrophil head-on, the neutrophil forms a thin tubelike protrusion around the bacterium, and the fully covered spirochete is drawn into the cell.

scholarly journals Development of a sensitive molecular diagnostic assay for detecting Borrelia burgdorferi DNA from the blood of Lyme disease patients by digital PCR

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0235372
Author(s):  
Srirupa Das ◽  
Denise Hammond-McKibben ◽  
Donna Guralski ◽  
Sandra Lobo ◽  
Paul N. Fiedler
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Digital Pcr ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Diagnostic Assay ◽  
Timely Manner ◽  
Blood Samples ◽  
Pcr Technology ◽  
Analytical Processing

Lyme disease patients would greatly benefit from a timely, sensitive, and specific molecular diagnostic test that can detect the causal agent Borrelia burgdorferi at the onset of symptoms. Currently available diagnostic methods recommended by the Centers for Disease Control and Prevention for Lyme disease involve indirect serological tests that rely on the detection of a host-antibody response, which often takes more than three weeks to develop. With this process, many positive cases are not detected within a timely manner, preventing a complete cure. In this study, we have developed a digital polymerase chain reaction (PCR) assay that detects Lyme disease on clinical presentation with a sensitivity two-fold higher than that of the currently available diagnostic methods, using a cohort of patient samples collected from the Lyme disease endemic state of Connecticut, USA, in 2016–2018. Digital PCR technology was chosen as it is more advanced and sensitive than other PCR techniques in detecting rare targets. The analytical detection sensitivity of this diagnostic assay is approximately three genome copies of B. burgdorferi. The paucity of spirochetes in the bloodstream of Lyme disease patients has hindered the clinical adoption of PCR-based diagnostic tests. However, this drawback was overcome by using a comparatively larger sample volume, applying pre-analytical processing to the blood samples, and implementing a pre-amplification step to enrich for B. burgdorferi-specific gene targets before the patient samples are analyzed via digital PCR technology. Pre-analytical processing of blood samples from acute patients revealed that the best sample type for Lyme disease detection is platelet-rich plasma rather than whole blood. If detected in a timely manner, Lyme disease can be completely cured, thus limiting antibiotic overuse and associated morbidities.

scholarly journals Evaluation of a New Culture Medium forBorrelia burgdorferi

2000 ◽  
Vol 38 (11) ◽  
pp. 4239-4241 ◽  
Author(s):  
Adriana R. Marques ◽  
Frida Stock ◽  
Vee Gill
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Culture Medium ◽  
Blood Samples ◽  
Growth Studies ◽  
Negative Growth

We evaluated the new MPM medium for the growth of Borrelia burgdorferi. All 18 blood samples from 17 patients with Lyme disease were negative. Growth studies showed that by day 4, most organisms in MPM were not viable. Our results reinforce the use of BSK medium as the primary choice for growing B. burgdorferi.

scholarly journals Tick Saliva Reduces Adherence and Area of Human Neutrophils

2004 ◽  
Vol 72 (5) ◽  
pp. 2989-2994 ◽  
Author(s):  
Ruth R. Montgomery ◽  
Denise Lusitani ◽  
Anne de Boisfleury Chevance ◽  
Stephen E. Malawista
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Polymorphonuclear Leukocyte ◽  
Natural Infection ◽  
Ixodid Ticks ◽  
Human Neutrophils ◽  
Tick Saliva ◽  
Anti Inflammatory ◽  
Β2 Integrins

ABSTRACT During natural infection with the agent of Lyme disease, Borrelia burgdorferi, spirochetes are delivered with vector saliva, which contains anti-inflammatory and antihemostatic activities. We show here that the saliva of ixodid ticks reduces polymorphonuclear leukocyte (PMN) adhesion via downregulation of β2-integrins and decreases the efficiency of PMN in the uptake and killing of spirochetes. Inhibition of integrin adhesion and signaling reduces anti-inflammatory functions of PMN. These effects may favor the initial survival of spirochetes in vivo.

scholarly journals Development of a sensitive molecular diagnostic assay for detecting Borrelia burgdorferi DNA from blood of Lyme disease patients by digital PCR

2020 ◽  
Author(s):  
Srirupa Das ◽  
Denise Hammond-McKibben ◽  
Donna Guralski ◽  
Sandra Lobo ◽  
Paul N. Fiedler
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Digital Pcr ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Sample Type ◽  
Diagnostic Assay ◽  
Blood Samples ◽  
Pcr Technology ◽  
Analytical Processing

AbstractLyme disease patients would benefit greatly from a timely, sensitive and specific molecular diagnostic test that can detect the causal agent, Borrelia burgdorferi, at the onset of symptoms. Currently available diagnostic methods recommended by the Centers for Disease Control and Prevention for Lyme disease, involve indirect serological tests that rely on the detection of a host-antibody response which often takes more than three weeks to develop. This results in non-detection of many genuine cases on a timely basis, preventing complete cure. In this study we have developed a digital PCR (polymerase chain reaction) assay that detects Lyme disease on clinical presentation at twice the sensitivity of the currently available diagnostic methods, using a cohort of patient samples collected from the Lyme disease endemic state of Connecticut, USA in 2016-2018. Digital PCR technology was chosen as it is more advanced and sensitive than other PCR techniques in detecting rare targets and the lower limit of detection of this diagnostic assay was found to be three genome copies of B. burgdorferi. The paucity of spirochetes in the bloodstream of Lyme disease patients that hinders the clinical adoption of PCR-based diagnostic tests, was overcome by using a comparatively larger sample volume, pre-analytical processing of blood samples and a pre-amplification step to enrich for B. burgdorferi-specific gene targets before using the digital PCR technology to analyze patient samples. Pre-analytical processing of blood samples from acute patients revealed that the best sample type for Lyme disease detection is platelet-rich plasma and not whole blood. If detected on time, Lyme disease can be cured completely limiting the overuse of antibiotics and associated morbidities.

scholarly journals Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

2021 ◽  
Vol 12 ◽  
Author(s):  
Sinan Muldur ◽  
Douangsone D. Vadysirisack ◽  
Sharan Ragunathan ◽  
Yalan Tang ◽  
Alonso Ricardo ◽  
...  
Keyword(s):  
Complement Activation ◽  
Ex Vivo ◽  
Human Neutrophils ◽  
Complement Factor ◽  
C5a Receptor ◽  
Cellular Immune Responses ◽  
Immunological Diseases ◽  
Microfluidic Assays ◽  
Cellular Immune ◽  
Potential Complement

Complement activation is key to anti-microbial defenses by directly acting on microbes and indirectly by triggering cellular immune responses. Complement activation may also contribute to the pathogenesis of numerous inflammatory and immunological diseases. Consequently, intense research focuses on developing therapeutics that block pathology-causing complement activation while preserving anti-microbial complement activities. However, the pace of research is slowed down significantly by the limitations of current tools for evaluating complement-targeting therapeutics. Moreover, the effects of potential therapeutic agents on innate immune cells, like neutrophils, are not fully understood. Here, we employ microfluidic assays and measure chemotaxis, phagocytosis, and swarming changes in human neutrophils ex vivo in response to various complement-targeting agents. We show that whereas complement factor 5 (C5) cleavage inhibitor eculizumab blocks all neutrophil anti-microbial functions, newer compounds like the C5 cleavage inhibitor RA101295 and C5a receptor antagonist avacopan inhibit chemotaxis and swarming while preserving neutrophil phagocytosis. These results highlight the utility of microfluidic neutrophil assays in evaluating potential complement-targeting therapeutics.

New ultrastructural findings about Borrelia burgdorferi by cryofixation, negative staining, thin sectioning and scanning techniques

1990 ◽  
Vol 48 (3) ◽  
pp. 582-583
Author(s):  
S. F. Hayes ◽  
M. D. Corwin ◽  
T. G. Schwan ◽  
D. W. Dorward ◽  
W. Burgdorfer
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Structural Characterization ◽  
Negative Staining ◽  
Low Speed ◽  
Thin Sectioning ◽  
Ultrastructural Findings

Characterization of Borrelia burgdorferi strains by means of negative staining EM has become an integral part of many studies related to the biology of the Lyme disease organism. However, relying solely upon negative staining to compare new isolates with prototype B31 or other borreliae is often unsatisfactory. To obtain more satisfactory results, we have relied upon a correlative approach encompassing a variety EM techniques, i.e., scanning for topographical features and cryotomy, negative staining and thin sectioning to provide a more complete structural characterization of B. burgdorferi.For characterization, isolates of B. burgdorferi were cultured in BSK II media from which they were removed by low speed centrifugation. The sedimented borrelia were carefully resuspended in stabilizing buffer so as to preserve their features for scanning and negative staining. Alternatively, others were prepared for conventional thin sectioning and for cryotomy using modified procedures. For thin sectioning, the fixative described by Ito, et al.

scholarly journals Surveillance of Ticks and Tick-Borne Pathogens in Suburban Natural Habitats of Central Maryland

2021 ◽  
Author(s):  
Matthew T Milholland ◽  
Lars Eisen ◽  
Robyn M Nadolny ◽  
Andrias Hojgaard ◽  
Erika T Machtinger ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Spotted Fever ◽  
Babesia Microti ◽  
Borrelia Burgdorferi Sensu Lato ◽  
Spotted Fever Group ◽  
Borrelia Miyamotoi ◽  
Natural Habitats ◽  
Questing Ticks ◽  
Tick Vectors

Abstract Lyme and other tick-borne diseases are increasing in the eastern United States and there is a lack of research on integrated strategies to control tick vectors. Here we present results of a study on tick-borne pathogens detected from tick vectors and rodent reservoirs from an ongoing 5-yr tick suppression study in the Lyme disease-endemic state of Maryland, where human-biting tick species, including Ixodes scapularis Say (Acari: Ixodidae) (the primary vector of Lyme disease spirochetes), are abundant. During the 2017 tick season, we collected 207 questing ticks and 602 ticks recovered from 327 mice (Peromyscus spp. (Rodentia: Cricetidae)), together with blood and ear tissue from the mice, at seven suburban parks in Howard County. Ticks were selectively tested for the presence of the causative agents of Lyme disease (Borrelia burgdorferi sensu lato [s.l.]), anaplasmosis (Anaplasma phagocytophilum), babesiosis (Babesia microti), ehrlichiosis (Ehrlichia ewingii, Ehrlichia chaffeensis, and ‘Panola Mountain’ Ehrlichia) and spotted fever group rickettsiosis (Rickettsia spp.). Peromyscus ear tissue and blood samples were tested for Bo. burgdorferi sensu stricto (s.s), A. phagocytophilum, Ba. microti, and Borrelia miyamotoi. We found 13.6% (15/110) of questing I. scapularis nymphs to be Bo. burgdorferi s.l. positive and 1.8% (2/110) were A. phagocytophilum positive among all sites. Borrelia burgdorferi s.s. was found in 71.1% (54/76) of I. scapularis nymphs removed from mice and 58.8% (194/330) of captured mice. Results from study on tick abundance and pathogen infection status in questing ticks, rodent reservoirs, and ticks feeding on Peromyscus spp. will aid efficacy evaluation of the integrated tick management measures being implemented.

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