Electrogenerated chemiluminescence (ECL) of water-soluble core/shell CdSe/ZnS quantum dots (QDs) coated with carboxylated polyethylene glycol polymers (“Qdot 625”) was investigated in aqueous solutions using 2-(dibutylamino)ethanol (DBAE) and tri-n-propylamine (TPrA) as ECL coreactants. In both cases, ECL emissions at glassy carbon (GC) electrode appeared at the same potential of ∼0.80 V vs. Ag/AgCl (3.0 M KCl), which was ∼200 and ∼150 mV more positive compared with the oxidation potentials for DBAE (∼+0.60 V vs. Ag/AgCl) and TPrA (∼+0.65 V vs. Ag/AgCl), respectively. The ECL intensity, however, was significantly affected by the type and the concentration of the ECL coreactant used as well as the nature of the working electrode. Under the present experimental conditions, ECL from DBAE was ∼17 times stronger than that from TPrA. The maximum ECL was obtained at GC electrode when [DBAE] ≈ 53 mM, where a ratio of 11 : 3 : 1 in ECL intensity was evaluated for GC, Au, and Pt electrodes, respectively. The ECL emission of the Qdot 625/DBAE system had an apparent peak value of ∼625 nm that matched well the fluorescence data. The QD as a label for ECL-based immunoassays of C-reactive protein (CRP) was realized by covalent binding of avidin on its surface, which allowed biotinylated anti-CRP to be attached and interacted with solution-phase CRP and the anti-CRP linked to micro-sized magnetic beads. The newly formed sandwich type aggregates were separated magnetically from the solution matrix, followed by the ECL generation at partially transparent Au nanoparticle-coated ITO electrode or Au/CD electrode in the presence of DBAE. Much stronger ECL responses were observed from the Au/CD electrode, at which a dynamic range of 1.0–10.0 μg mL⁻¹ CRP and a limit of detection of 1.0 μg mL⁻¹ CRP were obtained, respectively.

The geographical overlap of multiple Rickettsia and tick species coincides with the molecular detection of a variety of rickettsial agents in what may be novel tick hosts. However, little is known concerning transmissibility of rickettsial species by various tick hosts. To examine the vertical transmission potential between select tick and rickettsial species, two sympatric species of ticks, Dermacentor variabilis and Amblyomma maculatum, were exposed to five different rickettsial species, including Rickettsia rickettsii, Rickettsia parkeri, Rickettsia montanensis, Rickettsia amblyommatis, or flea-borne Rickettsia felis. Fitness-related metrics including engorgement weight, egg production index, nutrient index, and egg hatch percentage were then assessed. Subsamples of egg clutches and unfed larvae, nymphs, and adults for each cohort were assessed for transovarial and transstadial transmission of rickettsiae by qPCR. Rickettsial exposure had a minimal fitness effect in D. variabilis and transovarial transmission was observed for all groups except R. rickettsii. In contrast, rickettsial exposure negatively influenced A. maculatum fitness and transovarial transmission of rickettsiae was demonstrated only for R. amblyommatis- and R. parkeri-exposed ticks. Sustained maintenance of rickettsiae via transstadial transmission was diminished from F1 larvae to F1 adults in both tick species. The findings of this study suggest transovarial transmission specificity may not be tick species dependent, and sustained vertical transmission is not common.

Scientific analysis of the genus Rickettsia is undergoing a rapid period of change with the emergence of viable genetic tools. The development of these tools for the mutagenesis of pathogenic bacteria will permit forward genetic analysis of Rickettsia pathogenesis. Despite these advances, uncertainty still remains regarding the use of plasmids to study these bacteria in in vivo mammalian models of infection, namely, the potential for virulence changes associated with the presence of extrachromosomal DNA and nonselective persistence of plasmids in mammalian models of infection. Here, we describe the transformation of Rickettsia conorii Malish 7 with the plasmid pRam18dRGA[AmTrCh]. Transformed R. conorii stably maintains this plasmid in infected cell cultures, expresses the encoded fluorescent proteins, and exhibits growth kinetics in cell culture similar to those of nontransformed R. conorii . Using a well-established murine model of fatal Mediterranean spotted fever, we demonstrate that R. conorii (pRam18dRGA[AmTrCh]) elicits the same fatal outcomes in animals as its untransformed counterpart and, importantly, maintains the plasmid throughout infection in the absence of selective antibiotic pressure. Interestingly, plasmid-transformed R. conorii was readily observed both in endothelial cells and within circulating leukocytes. Together, our data demonstrate that the presence of an extrachromosomal DNA element in a pathogenic rickettsial species does not affect either in vitro proliferation or in vivo infectivity in models of disease and that plasmids such as pRam18dRGA[AmTrCh] are valuable tools for the further genetic manipulation of pathogenic rickettsiae.

The Gram-negative obligate intracellular bacterium Rickettsia parkeri is an emerging tick-borne human pathogen. Recently, R. parkeri Sca2 and RickA have been implicated in adherence and actin-based motility in vertebrate host cell infection models; however, the rickettsia-derived factors essential to tick infection are unknown. Using R. parkeri mutants lacking functional Sca2 or RickA to compare actin polymerization, replication, and cell-to-cell spread in vitro , similar phenotypes in tick and mammalian cells were observed. Specifically, actin polymerization in cultured tick cells is controlled by the two separate proteins in a time-dependent manner. To assess the role of Sca2 and RickA in dissemination in the tick host, Rickettsia -free Amblyomma maculatum , the natural vector of R. parkeri , was exposed to wild-type, R. parkeri rickA :: tn , or R. parkeri sca2 :: tn bacteria, and individual tick tissues, including salivary glands, midguts, ovaries, and hemolymph, were analyzed at 12 h and after continued bloodmeal acquisition for 3 or 7 days postexposure. Initially, ticks exposed to wild-type R. parkeri had the highest rickettsial load across all organs; however, rickettsial loads decreased and wild-type rickettsiae were cleared from the ovaries at 7 days postexposure. In contrast, ticks exposed to R. parkeri rickA :: tn or R. parkeri sca2 :: tn had comparatively lower rickettsial loads, but bacteria persisted in all organs for 7 days. These data suggest that while RickA and Sca2 function in actin polymerization in tick cells, the absence of these proteins did not change dissemination patterns within the tick vector.

Tick vectors are capable of transmitting several rickettsial species to vertebrate hosts, resulting in various levels of disease. Studies have demonstrated the transmissibility of both rickettsial pathogens and novel Rickettsia species or strains with unknown pathogenicity to vertebrate hosts during tick blood meal acquisition; however, the quantitative nature of transmission remains unknown.

The tick-borne spirochete,Borrelia miyamotoi, is an emerging pathogen of public health significance. CurrentB.miyamotoiserodiagnostic testing depends on reactivity against GlpQ which is not highly sensitive on acute phase serum samples. Additionally, anti-B.miyamotoiantibodies can cross-react with C6 antigen testing forB.burgdorferi, the causative agent of Lyme disease, underscoring the need for improved serological assays that produce accurate diagnostic results. We performed an immunoproteomics analysis ofB.miyamotoiproteins to identify novel serodiagnostic antigens. Sera from mice infected withB.miyamotoiby subcutaneous inoculation or tick bite were collected for immunoblotting againstB.miyamotoimembrane-associated proteins separated by 2-dimensional electrophoresis (2DE). In total, 88 proteins in 40 2DE immunoreactive spots were identified via mass spectrometry. Multiple variable large proteins (Vlps) and a putative lipoprotein were among those identified and analyzed. Reactivity of anti-B.miyamotoisera against recombinant Vlps and the putative lipoprotein confirmed their immunogenicity. Mouse anti-B.burgdorferiserum was cross-reactive to all recombinant Vlps, but not against the putative lipoprotein by IgG. Furthermore, antibodies against the recombinant putative lipoprotein were present in serum from aB.miyamotoi-infected human patient, but not a Lyme disease patient. Results presented here provide a comprehensive profile ofB.miyamotoiantigens that induce the host immune response and identify a putative lipoprotein as a potentially specific antigen forB.miyamotoiserodetection.