• Detection of species-specific DNA sequences of Borrelia burgdorferi in infected humans, animal reservoirs, and ixodid tick vectors

      Malloy, Diane Catherine; Nauman, Robert K. (1992)
      Segments of the ospA gene that encode hydrophobic regions of the outer membrane protein, OspA, of Borrelia burgdorferi strain B31 were synthesized for use as oligonucleotide primers in the polymerase chain reaction (PCR). These oligonucleotide primers flank a 309-base-pair segment within the ospA gene. Optimal amplification conditions were achieved in a reaction mixture containing 0.2 uM of each oligonucleotide primer and 2 mMMgCl{dollar}\sb2.{dollar} Dimethyl sulfoxide at a concentration of 10% or higher was found to inhibit amplification and gelatin had no effect at concentrations below 100 ug/ml, and slight inhibition was seen at concentrations higher than 100 ug/ml. After 30 cycles of amplification under optimal conditions, the target fragment could be detected by agarose gel electrophoresis or dot hybridization with a {dollar}\sp{lcub}32{rcub}{dollar}P- or digoxigenin-labeled probe. This segment was amplified in all strains of B. burgdorferi, but it was not detected in other bacterial species. The sensitivity of PCR for the detection of B. burgdorferi in clinical samples was evaluated by seeding blood and urine specimens with B. burgdorferi and subjecting them to amplification. Ten organisms per ml of blood or urine could be detected using PCR with dot hybridization detection. In a blinded study of Lyme disease patients, the OspA PCR was positive in 31% of patients who were early in disease and who had not received oral antibiotic therapy. No patient who had received antibiotics was positive in the PCR. Blood and urine specimens were obtained from canines with clinical and serologic evidence of Lyme disease and subjected to PCR analysis. Of 17 clinical specimens from 15 canines, one blood specimen showed reactivity in the PCR. Two of 32 cerebrospinal fluid specimens from suspected neuroborreliosis patients showed reactivity in the PCR. B. burgdorferi could be detected optimally in tissue only after DNA extraction. Nine of ten mice from a highly endemic Lyme disease area in Wisconsin showed reactivity in the PCR when DNA extracted from heart, kidney, or bladder was used as the target. Two of five punch biopsy tissue samples from skin lesions from suspected Lyme disease patients showed reactivity in the PCR. Of all tissues studied, one yielded a positive spirochete stain and all were negative by immunoperoxidase staining with a polyclonal antibody to B. burgdorferi. The conclusion of this study is that PCR can detect and identify B. burgdorferi in clinical samples from Lyme disease with greater sensitivity than any other currently available method and that this tool can be used to detect the spirochete in tick and animal reservoirs.
    • XIAP-p47 Pairing Activates the Immune Deficiency Pathway in the Lyme Disease Tick Ixodes scapularis

      McClure Carroll, Erin; Pedra, Joao H. F. (2019)
      Globally, vector-borne diseases account for 17% of all infectious diseases. Most vectors are blood-feeding arthropods, which transmit bacterial, viral, and parasitic diseases to humans and animals. The tick Ixodes scapularis transmits seven pathogens, including Borrelia burgdorferi, the agent of Lyme disease. Lyme disease is the most important vector-borne disease in the United States and causes an estimated 329,000 infections annually. Best described in the model organism Drosophila melanogaster, the arthropod immune deficiency (IMD) pathway responds to microbial infection through activation of Relish, a nuclear factor (NF)-κB family transcription factor. In I. scapularis ticks, the E3 ubiquitin ligase X-linked inhibitor of apoptosis (XIAP) regulates the IMD pathway through ubiquitylation. Yet, the tick genome notably lacks homologs to genes encoding key IMD pathway proteins as described in Drosophila. How XIAP activates the IMD pathway in response to microbial infection is poorly characterized and targets of XIAP-mediated ubiquitylation remain unknown. In this study, we identified the XIAP enzymatic substrate p47 as a positive regulator of the I. scapularis IMD network. XIAP polyubiquitylates p47 in a lysine (K)63-dependent manner and interacts with the ubiquitin-like (UBX) domain of p47. p47 also binds to Kenny (IKKγ/NF-κB essential modulator [NEMO]), the regulatory subunit of the inhibitor of NF-κB kinase (IKK) complex. Replacement of the amino acid lysine with arginine in the p47 linker region completely abrogated molecular interactions with Kenny. Furthermore, reduction of p47 transcription levels through RNA interference in I. scapularis limited Kenny accumulation, reduced phosphorylation of IKKβ (IRD5), and impaired cleavage of the NF-κB molecule Relish. Accordingly, disruption of p47 expression increased microbial colonization of the tick-transmitted spirochete B. burgdorferi and the rickettsial agent Anaplasma phagocytophilum. In summary, we demonstrated that XIAP ubiquitylates p47 in a K63-dependent manner, culminating in Relish activation and antimicrobial responses. Manipulating immune signaling cascades in I. scapularis may lead to innovative approaches to reducing the burden of tick-borne diseases.