Improved Culture Conditions for the Growth and Detection of Borrelia from Human Serum

In this report we present a method to cultivate Borrelia spirochetes from human serum samples with high efficiency. This method incorporates improved sample collection, optimization of culture media and use of matrix protein. The method was first optimized utilizing Borrelia laboratory strains, and later by demonstrating growth of Borrelia from sera from fifty seropositive Lyme disease patients followed by another cohort of 72 Lyme disease patients, all of whom satisfied the strict CDC surveillance case definition for Lyme disease. The procedure resulted in positive cultures in 47% at 6 days and 94% at week 16. Negative controls included 48 cases. The positive identification of Borrelia was performed by immunostaining, PCR, and direct DNA sequencing

Filarial Nematode Infection in Ixodes scapularis Ticks Collected from Southern Connecticut

It was recently demonstrated that the lone star tick Amblyomma americanum could harbor filarial nematodes within the genus Acanthocheilonema. In this study, Ixodes scapularis (deer) ticks collected from Southern Connecticut were evaluated for their potential to harbor filarial nematodes. Non-engorged nymphal and adult stage Ixodes scapularis ticks were collected in Southern Connecticut using the standard drag method. In situ hybridization with filarial nematode specific sequences demonstrated the presence of filarial nematodes in Ixodes ticks. Filarial nematode specific DNA sequences were amplified and confirmed by direct sequencing in Ixodes nymphal and adult ticks using either general filarial nematode or Onchocercidae family specific PCR primers. Phylogenetic analysis of the 12S rDNA gene sequence indicated that the filarial nematode infecting Ixodes scapularis ticks is most closely related to the species found in Amblyoma americanum ticks and belongs to the genus of Acanthocheilonema. Our data also demonstrated that infection rate of these filarial nematode in Ixodes ticks is relatively high (about 22% and 30% in nymphal and adult Ixodes ticks, respectively). In summary, the results from our studies demonstrated that filarial nematode infection was found in Ixodes ticks similar to what has been found in Amblyomma americanum ticks

Characterization of Biofilm Formation by Borrelia burgdorferi In Vitro

Borrelia burgdorferi, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that Borrelia burgdorferi aggregate formation dramatically changes the in vitro response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied Borrelia burgdorferi for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS) matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that Borrelia burgdorferi is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that Borrelia burgdorferi is capable of forming biofilm in vitro. Biofilm formation by Borrelia species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells

Filarial Nematode Infection in Ixodes scapularis Ticks Collected from Southern Connecticut

It was recently demonstrated that the lone star tick Amblyomma americanum could harbor filarial nematodes within the genus Acanthocheilonema. In this study, Ixodes scapularis (deer) ticks collected from Southern Connecticut were evaluated for their potential to harbor filarial nematodes. Non-engorged nymphal and adult stage Ixodes scapularis ticks were collected in Southern Connecticut using the standard drag method. In situ hybridization with filarial nematode specific sequences demonstrated the presence of filarial nematodes in Ixodes ticks. Filarial nematode specific DNA sequences were amplified and confirmed by direct sequencing in Ixodes nymphal and adult ticks using either general filarial nematode or Onchocercidae family specific PCR primers. Phylogenetic analysis of the 12S rDNA gene sequence indicated that the filarial nematode infecting Ixodes scapularis ticks is most closely related to the species found in Amblyoma americanum ticks and belongs to the genus of Acanthocheilonema. Our data also demonstrated that infection rate of these filarial nematode in Ixodes ticks is relatively high (about 22% and 30% in nymphal and adult Ixodes ticks, respectively). In summary, the results from our studies demonstrated that filarial nematode infection was found in Ixodes ticks similar to what has been found in Amblyomma americanum ticks

Characterization of Biofilm Formation by <em>Borrelia burgdorferi In Vitro</em>

<div><p><em>Borrelia burgdorferi</em>, the causative agent of Lyme disease, has long been known to be capable of forming aggregates and colonies. It was recently demonstrated that <em>Borrelia burgdorferi</em> aggregate formation dramatically changes the <em>in vitro</em> response to hostile environments by this pathogen. In this study, we investigated the hypothesis that these aggregates are indeed biofilms, structures whose resistance to unfavorable conditions are well documented. We studied <em>Borrelia burgdorferi</em> for several known hallmark features of biofilm, including structural rearrangements in the aggregates, variations in development on various substrate matrices and secretion of a protective extracellular polymeric substance (EPS) matrix using several modes of microscopic, cell and molecular biology techniques. The atomic force microscopic results provided evidence that multilevel rearrangements take place at different stages of aggregate development, producing a complex, continuously rearranging structure. Our results also demonstrated that <em>Borrelia burgdorferi</em> is capable of developing aggregates on different abiotic and biotic substrates, and is also capable of forming floating aggregates. Analyzing the extracellular substance of the aggregates for potential exopolysaccharides revealed the existence of both sulfated and non-sulfated/carboxylated substrates, predominately composed of an alginate with calcium and extracellular DNA present. In summary, we have found substantial evidence that <em>Borrelia burgdorferi</em> is capable of forming biofilm <em>in vitro.</em> Biofilm formation by <em>Borrelia</em> species might play an important role in their survival in diverse environmental conditions by providing refuge to individual cells.</p> </div

<i>Borrelia burgdorferi</i> B31 strain large aggregate surrounded by individual spirochetes and several small aggregates stained with the calcium-detecting stain Alizarin.

<p>Red coloration indicates presence of calcium, by differential interference contrast (Panel A) and dark field microscopy (Panel B). White arrows indicate unstained spirochetes and small aggregates. 400× magnification.</p

Representative images of <i>Borrelia burgdorferi</i> B31 strain aggregates in the early (1^st column, 0 to 2 day), middle (2^nd column, 3 to 6 days) and late (3^rd column, 7 to 21 days) stages of development, observed with dark field (A, B, C – 400× magnification); differential interference contrast (D, E, F - 400× magnification); and FITC-band epifluorescence (G, H, I – 400× magnification).

<p>D, F, and I are extended depth of field composites.</p

Representative image showing Spicer & Meyer aldehyde fuchsine-Alcian blue sequential staining pattern of two <i>Borrelia burgdorferi</i> B31 strain aggregates by differential interference contrast (A and B) and dark field microscopy (C and D).

<p>Fuchsia coloration is indicative of weakly acidic sulfomucin; purple coloration indicates strongly acidic sulfomucins and/or sulfated proteoglycans; blue coloration indicates non-sulfated, carboxylated mucins. 500× magnification.</p

Immunohistochemical staining of a collagen-embedded <i>Borrelia burgdorferi</i> B31 aggregates (Panels A–C and G–I) and individual spirochetal cells (Panels D–F) for alginate (red staining; Panels B andI) and <i>Borrelia</i> antigen (green staining; Panels A,D,G) expression using fluorescent microscopy (see methods for detailed protocol).

<p>Panel H shows the lack of red staining in the absence of the primary antibody for alginate. Panels C, F and I show DAPI - DNA counterstain images. 400× magnification.</p

Three-dimensional AFM images of <i>Borrelia burgdorferi</i> B31 strain early aggregates on agarose substrate.

<p>The preparation of <i>Borrelia burgdorferi</i> cells on mica is described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048277#s4" target="_blank">Materials and Methods</a>. The sample was scanned at 0.3 Hz using contact mode. A: The original AFM image produced with NanoRule© software. B: The AFM dataset was converted to a 3D mesh via a custom meshing utility, cleaned with MeshLab open-source software, then imported to Adobe Photoshop and false-color hand-painted with 3D painting tools. White: spirochete bodies; blue: potential EPS matrix; purple: protrusions; and yellow: small round bodies.</p