This work was partially supported by the Natural Science and Engineering Research Council of Canada and the Canadian Lyme disease Foundation (AB and VL), the Swedish Research Council (SB, M-LF and IN), TA&#268;R GAMA 2 project - &#34;Support of application potential verification 2.0 at the Biology Centre CAS&#34; (TP01010022) (MG and NR), and by a grant from Ministry of Health of the Czech Republic NV19-05-00191 (MG and NR). We thank S. Vranjes (2021, Rudenko lab) for the image in Figure 4 and J. Thomas-Ebbett (2021, Lloyd lab) for the images in Figure 5. We thank all researchers who have contributed to the field and apologize to those whose work we were not able to cite due to space limitations.

No conflicts of interest were declared.

The laboratory culture of bacteria is the springboard for research. The profound advantage conferred by the ability to culture is exemplified by the struggle, over more than a century culminating only recently in success, to culture Treponema pallidum, the spirochete that is the etiological agent of syphilis44. Borrelia spirochetes are also challenging to culture, but culture is possible23,24,34,44,45,46,47,48,49.
Borrelia cells are fastidious and each species and strain, and even isolate differs, both genetically and through adaptation to the host from which they have been isolated51,52,53,54,55. When making media, the use of appropriate ingredients make an enormous difference in the vigor of Borrelia culture or even the viability of the culture. The&#160;use of freshly prepared, highly enriched, and properly stored medium is essential to promote bacterial growth and is a well-established requirement for bacterial growth. For Borrelia, maintaining anaerobic or microaerobic conditions is required for good growth. This can be achieved most simply by filling tubes to the top, so there is little to no air remaining in the tube and growing the cultures without agitation; more stringent control of oxygen and carbon dioxide may be required for gene expression and related studies26,27,28,29,30. The addition of antibiotics to the bacterial culture medium may seem counter-intuitive; the use of low doses of antibiotics in the Borrelia medium is to discourage the growth of faster-growing bacteria. If the culture is inoculated with a pure Borrelia isolate, the addition of antibiotics is not needed; however&#160;the use of added antibiotics is essential to discourage overgrowth from competing bacteria when inoculating with clinical or environmental samples. BSK medium can also be supplemented with 3%-7% gelatin. This is needed for the growth of RF Borrelia species21. The use of BSK or MKP for the growth of Borrelia burgdorferi sensu lato species depends on the source of the isolate. Both media support the growth of established cultures, but MKP has been found to perform better when seeding new cultures with low numbers of Borrelia cells, such as occurs with clinical or environmental isolates23,24. This medium also allows the culture of B. miyamotoi56,57,58. Ultimately, there is an element of art to the science of growing fastidious microbes, but it is possible with care and persistence.
The culture of Borrelia underlies advances in biomedical research. The culture of Borrelia has allowed determination of the full genome and proteome of many Borrelia species, which has permitted researchers to start to unravel the evolution and biology of these spirochetes54,58,59. Similarly, access to pure cultures of Borrelia allows the understanding of the many complex interactions of Borrelia with the mammalian host and arthropod vector61,62,63,64,65, including interactions with the host immune system11,65, allows mechanisms of transmission to be inferred29,66, and provides the key investigative tool to distinguish culturable, hence necessarily living, cells from cellular debris, a distinction that is critical in unraveling the mechanisms of pathogenesis as well as effective treatment regimens8,67,68,69,70,71. While the in vitro Borrelia culture can take several weeks before spirochetes are sufficiently abundant for detection, limiting clinical diagnostic applications, fundamental understanding of Borrelia biology, genomics, host-pathogen interactions, and many more applications has relied on the ability to isolate pure Borrelia cultures from environmental isolates, and to amplify those isolates through culture to produce sufficient material for biochemical and genetic analysis. There is a societal push for rapid response to infections to support rapid diagnosis and treatment. The other component of this need, however, is the biomedical and biological research needed for a solid foundation upon which to appropriately and successfully treat and prevent disease. While challenging, in vitro cultivation of Borrelia spirochetes is possible and can support these needs.

Borrelia is a genus of spirochete bacteria that encompasses three major clades: the Lyme borreliosis (LB) group, the relapsing fever (RF) group, and a less well-characterized group seemingly restricted to reptiles. Borrelia taxonomy is in flux with the advent of molecular methodologies that allow genome and proteome comparisons, as is true of most other taxonomic groups1,2,3,4,5,6,7. The LB group (also called the Lyme disease group) has traditionally been termed Borrelia burgdorferi sensu lato after its best-characterized member Borrelia burgdorferi sensu stricto.&#160;This paper uses the currently most widely used terminology: the LB, the RF, and the reptile-associated group, and describes the culture protocols for the LB and the RF groups.
As would be expected for a member of the family Spirochaetaceae, Borrelia can adopt the distinctively long and thin spiral shape, typically 20-30 &#181;m long and 0.2-0.3 &#181;m wide. However, Borrelia cells are highly pleomorphic and can adopt many other shapes in both culture and in vivo1,8 as a result of their complex cellular and genetic structure. In its spirochetal form, the planar sine wave morphology results from its axial endoflagella rotating in the periplasmic space between the inner and outer membranes. This structure enables the cells to be highly motile, with the outer membrane containing proteins that enable the cell to interact with host tissues9,10. The expression of the outer membrane proteins is tightly regulated and affect not only host tissue invasion but also interaction with the host immune system11. This complex gene expression allows the Borrelia cells to shuttle between the very different environments of vertebrate hosts and invertebrate vectors. The genome of Borrelia is unusual among prokaryotes, consisting of a linear rather than a circular chromosome. In addition to the linear chromosome, Borrelia species contain 7-21 plasmids, some linear and some circular. The plasmids harbor the majority of genes required for host adaptation and virulence, and the circular plasmids derived from prophages are thought to be responsible for the majority of horizontal gene flow between spirochetal cells12,13. Consistent with a role in host adaptation, some, possibly many or all, members of the Lyme borreliosis group lose plasmids in culture14. The best studied &#34;lab adapted&#34; strain of B. burgdorferi, B31, has only seven of the nine plasmids found in wild isolates of this species15. Similarly, B. garinii loses plasmids in culture16. Some studies have shown that RF species and B. miyamotoi retain plasmids when cultured14,17, but recent work demonstrates altered plasmids and infectivity with long-term in vitro cultivation18.
Culture-based methods remain the gold standard for the laboratory detection of bacterial infections, for both research and clinical work14,17. The culture of pathogens from bodily fluids or tissues directly detects replicating pathogens and provides source material for research14,17. This protocol demonstrates the methodology and recipes required to successfully culture the spirochetes of the LB group as well as RF Borrelia and B. miyamotoi. The basic medium for growing Borrelia spirochetes is the Barbour-Stoenner-Kelly medium (BSK-II or the commercially available BSK-H), with or without antibiotics to reduce the growth of contaminating prokaryotes. This medium was adapted from a medium originally used to support RF Borrelia19, further modified by Stoenner20 and then by Barbour21. Many modifications have since been developed, each having effects on bacterial physiology that can affect growth, infectivity, and pathogenicity22. This medium reliably supports the growth of spirochetes in established cultures and has been used to isolate spirochetes from tick, mammal, and clinical samples23. The more recently developed variation, modified Kelly-Pettenkofer (MKP) medium, can provide better isolation success, morphology, and motility when isolating new Borrelia isolates from environmental samples, when the number of spirochetes present in the sample available to seed the culture is low23,24. In all cases, the success of cultivation is critically dependent on the freshly prepared medium and the use of appropriate ingredients; not all commercial ingredients produce high-quality medium. Inoculated cultures can be conveniently incubated without shaking in a conventional 32-34 &#176;C incubator in the presence of a small amount of residual ambient oxygen. Borrelia spirochetes are anaerobes but are exposed in nature to fluctuations in oxygen and carbon dioxide concentrations and respond with changes in gene expression26,27,28,29. Thus, gene expression, growth, and other metabolic studies should control for oxygen and carbon dioxide levels with the use of an oxygen-controlled incubator or anaerobic chamber. In culture, cultures are checked weekly, or more often, for the presence of spirochetes with either darkfield microscopy or phase contrast microscopy. Culture smears can be stained with either silver stains, immunohistochemistry, or through the use of fluorescently tagged strains29,30. PCR followed by DNA sequencing is a sensitive and specific method to detect and genetically identify or confirm the Borrelia species30,31,32,33.
Many minor variations on BSK-II exist, and some are commercially available. The protocol described here in section 1 has been adapted from Barbour (1984)21. Liquid MKP medium is a more recently developed medium and is described in section 2. It is prepared according to a previously reported protocol33,34, which, similarly to BSK medium, consists of two steps: preparation of basic medium and preparation of complete medium. Borrelia culture medium can be prepared with or without antibiotics, as described in section 3; the antibiotics act to reduce contaminating bacteria introduced when inoculating with clinical or environmental samples, as described in section 4; if inoculating with pure Borrelia sp. culture, antibiotics may not be needed. Making long-term Borrelia stocks is often important, and a protocol for doing so is described in section 5. Section 6 describes using these media to isolate pure Borrelia sensu lato clones from clinical or environmental samples. There are a number of approaches possible36; below is one found to be effective. The plating medium used in this protocol is a modification of BSK-II plating medium37&#160;and MKP medium34 (with rabbit serum increased to 10%38).

<table><tbody><tr><td>1.7 mL tubes</td><td>VWR</td><td>87003-294</td><td>Standard item - any supplier will do</td></tr><tr><td>0.2&amp;nbsp;&amp;micro;m Sterile syringe filter&amp;nbsp;</td><td>VWR</td><td>28145-501</td><td>Standard item - any supplier will do</td></tr><tr><td>10 &amp;micro;L barrier pipette tip</td><td>Neptune</td><td>BT10XLS3</td><td>Standard item - any supplier will do</td></tr><tr><td>10 mL Serological pipettes&amp;nbsp;</td><td>Celltreat</td><td>229011B</td><td>Standard item - any supplier will do</td></tr><tr><td>1000 &amp;micro;L barrier pipette tip</td><td>Neptune</td><td>BT1000.96</td><td>Standard item - any supplier will do</td></tr><tr><td>15 mL tube</td><td>Celltreat</td><td>188261</td><td>Standard item - any supplier will do</td></tr><tr><td>20 &amp;micro;L barrier pipette tip</td><td>Neptune</td><td>BT20</td><td>Standard item - any supplier will do</td></tr><tr><td>20 mL Sterile syringe&amp;nbsp;</td><td>BD</td><td>309661</td><td>Standard item - any supplier will do</td></tr><tr><td>200 &amp;micro;L barrier pipette tip</td><td>Neptune</td><td>BT200</td><td>Standard item - any supplier will do</td></tr><tr><td>25 mL Screw Cap Culture Tubes</td><td>Fisher Scientific</td><td>14-933C</td><td>Standard item - any supplier will do</td></tr><tr><td>25 mL Serological pipettes</td><td>Celltreat</td><td>229025B</td><td>Standard item - any supplier will do</td></tr><tr><td>3 mL Sterile syringe</td><td>BD</td><td>309657</td><td>Standard item - any supplier will do</td></tr><tr><td>35% BSA&amp;nbsp;</td><td>Sigma</td><td>A-7409</td><td>Source is important - see note</td></tr><tr><td>5 mL Serological pipettes&amp;nbsp;</td><td>Celltreat</td><td>229006B</td><td>Standard item - any supplier will do</td></tr><tr><td>50 mL tube</td><td>Celltreat</td><td>229421</td><td>Standard item - any supplier will do</td></tr><tr><td>6.5 ml MKP glass tubes&amp;nbsp;</td><td>Schott</td><td>Schott Nr. 26 135 115</td><td>Standard item - any supplier will do</td></tr><tr><td>Amikacine</td><td>Sigma</td><td>PHR1654</td><td>Standard item - any supplier will do</td></tr><tr><td>Amphotericin B</td><td>Sigma</td><td>A9528-100MG</td><td>Standard item - any supplier will do</td></tr><tr><td>Bactrim/rimethoprim/sulfamethoxazole</td><td>Sigma</td><td>PHR1126-1G</td><td>Standard item - any supplier will do</td></tr><tr><td>BBL Brucella broth&amp;nbsp;</td><td>BD</td><td>211088</td><td>Standard item - any supplier will do</td></tr><tr><td>Biosafety Cabinet</td><td>Labconco</td><td>302419100</td><td>Standard item - any supplier will do</td></tr><tr><td>Blood collection tubes (yellow top - ACD)</td><td>Fisher Scientific</td><td>BD&amp;nbsp;Vacutainer Glass Blood Collection Tubes with Acid Citrate Dextrose (ACD)</td><td>Standard item - any supplier will do</td></tr><tr><td>BSK-H Medium [w 6% Rabbit serum]&amp;nbsp;</td><td>Darlynn biologicals</td><td>BB83-500</td><td>Standard item - any supplier will do</td></tr><tr><td>centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>model 5430</td><td>Standard item - any supplier will do</td></tr><tr><td>Citric acid TrisodiumSaltDihydrate</td><td>Sigma</td><td>C-8532 100 g</td><td>Standard item - any supplier will do</td></tr><tr><td>CMRL</td><td>Gibco BRL</td><td>21540 500 mL</td><td>Standard item - any supplier will do</td></tr><tr><td>CMRL-1066</td><td>Gibco</td><td>21-510-018</td><td>Standard item - any supplier will do</td></tr><tr><td>Cryogenic Tubes (Nalgene)</td><td>Fisher Scientific</td><td>5000-0020</td><td>Standard item - any supplier will do</td></tr><tr><td>Deep Petri with stacking ring 100 mm &amp;times; 25 mm</td><td>Sigma</td><td>P7741</td><td>Standard item - any supplier will do</td></tr><tr><td>Digital Incubator</td><td>VWR</td><td>model 1545</td><td>Standard item - any supplier will do</td></tr><tr><td>DMSO</td><td>ThermoFisher</td><td>D12345</td><td>Standard item - any supplier will do</td></tr><tr><td>Filters for filter sterilization</td><td>Millipore 0.22&amp;mu;m GPExpressPLUS Membrane</td><td>SCGPU05RE</td><td>Standard item - any supplier will do</td></tr><tr><td>Gelatin</td><td>Difco BD</td><td>214340 500 g</td><td>Standard item - any supplier will do</td></tr><tr><td>Glass Culture Tubes</td><td>Fisher Scientific</td><td>99449-20</td><td>Standard item - any supplier will do</td></tr><tr><td>Glucose</td><td>Sigma</td><td>G-7021 1 kg</td><td>Standard item - any supplier will do</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td>Standard item - any supplier will do</td></tr><tr><td>Hemafuge (Hematocrit &amp;amp; Immuno hematology centrifuge )</td><td>Labwissen</td><td>Model 3220</td><td>Standard item - any supplier will do</td></tr><tr><td>HEPES</td><td>Sigma&amp;nbsp;</td><td>H-3784 100 g</td><td>Standard item - any supplier will do</td></tr><tr><td>N-acetylglucoseamine</td><td>Sigma&amp;nbsp;</td><td>A-3286 25 g</td><td>Standard item - any supplier will do</td></tr><tr><td>Neopeptone</td><td>Difco&amp;nbsp; BD</td><td>211681 500 g</td><td>Standard item - any supplier will do</td></tr><tr><td>Neubauer Hematocytometer</td><td>Sigma&amp;nbsp;</td><td>Z359629</td><td>Standard item - any supplier will do</td></tr><tr><td>Phase contrast microscope&amp;nbsp;</td><td>Leitz</td><td></td><td>Standard item - any supplier will do</td></tr><tr><td>Phosphomycin</td><td>Sigma</td><td>P5396-1G</td><td>Standard item - any supplier will do</td></tr><tr><td>Phosphomycine</td><td>Sigma</td><td>P5396</td><td>Standard item - any supplier will do</td></tr><tr><td>Pipetboy</td><td>Integra</td><td></td><td>Standard item - any supplier will do</td></tr><tr><td>Precision Standard Balance</td><td>OHAUS</td><td>model TS200S</td><td>Standard item - any supplier will do</td></tr><tr><td>Pyruvic acid (Na salt)</td><td>Sigma</td><td>P-8574 25 g</td><td>Standard item - any supplier will do</td></tr><tr><td>Rabbit Serum&amp;nbsp;</td><td>Gibco</td><td>16-120-032</td><td>Source is important&amp;nbsp;</td></tr><tr><td>Rabbit Serum&amp;nbsp;</td><td>Sigma</td><td>R-4505&amp;nbsp; 100 mL</td><td>Source is important&amp;nbsp;</td></tr><tr><td>Rifampicin</td><td>Sigma</td><td>R3501-1G</td><td>Standard item - any supplier will do</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S-5761&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 500 g</td><td>Standard item - any supplier will do</td></tr><tr><td>Sufametaxazole&amp;nbsp;</td><td>Sigma</td><td>PHR1126</td><td>Standard item - any supplier will do</td></tr><tr><td>TC Yeastolate</td><td>Difco&amp;nbsp; BD</td><td>255752 100 g</td><td>Standard item - any supplier will do</td></tr><tr><td>Transfer Pipettes</td><td>VWR</td><td>470225-044</td><td>Standard item - any supplier will do</td></tr><tr><td>Trimethoprim</td><td>Sigma</td><td>PHR1056</td><td>Standard item - any supplier will do</td></tr></tbody></table>

cultivation methods of spirochetes from borrelia burgdorferi sensu lato complex and relapsing fever borrelia

The Borrelia consists of three groups of species, those of the Lyme borreliosis (LB) group, also known as B. burgdorferi sensu lato (s.l.) and recently reclassified into Borreliella, the relapsing fever (RF) group Borrelia, and a third reptile-associated group of spirochetes. Culture-based methods remain the gold standard for the laboratory detection of bacterial infections for both research and clinical work, as the culture of pathogens from bodily fluids or tissues directly detects replicating pathogens and provides source material for research. Borrelia and&#160;Borreliella spirochetes&#160;are fastidious and slow growing, and thus are not commonly cultured for clinical purposes; however, culture is necessary for research. This protocol demonstrates the methodology and recipes required to successfully culture LB and RF spirochetes, including all recognized species from B. burgdorferi s.l. complex including&#160;B. afzelii, B. americana, B. andersonii, B. bavariensis, B. bissettii/bissettiae, B. burgdorferi sensu stricto&#160;(s.s.), B. californiensis, B. carolinensis, B. chilensis, B. finlandensis, B. garinii, B. japonica, B. kurtenbachii, B. lanei, B. lusitaniae, B. maritima, B. mayonii, B. spielmanii, B. tanukii, B. turdi, B. sinica, B. valaisiana, B. yangtzensis, and RFspirochetes, B. anserina, B. coriaceae, B. crocidurae, B. duttonii, B. hermsii, B. hispanica, B. persica, B. recurrentis, and B. miyamotoi. The basic medium for growing LB and RF spirochetes is the Barbour-Stoenner-Kelly (BSK-II or BSK-H) medium, which reliably supports the growth of spirochetes in established cultures. To be able to grow newly isolated Borrelia isolates from tick- or host-derived samples where the initial spirochete number is low in the inoculum, modified Kelly-Pettenkofer (MKP) medium is preferred. This medium also supports the growth of B. miyamotoi. The success of the cultivation of RF spirochetes also depends critically on the quality of ingredients.

Borrelia culture media BSK and MKP, and variants, are rich culture media with ingredients that need to be prepared and sterilized sequentially. When correctly prepared, BSK medium should be red-orange and clear (Figure 1). Turbidity and precipitation that persist after warming indicate problematic ingredients, medium production, or contamination; such medium is best discarded. If gelatin is added to BSK and with MKP, the medium will be gelatinous when refrigerated; upon warming, it will be liquid, although slightly viscous.
The growth of pure Borrelia cultures will not change the turbidity of the medium. However, the growth of bacterial cultures, Borrelia, as well as contaminants, will change medium color as a result of the pH indicator. Overgrowth by other bacteria will produce turbidity and clumped materials (Figure 2).
Culture growth can be monitored&#160;by phase contrast or darkfield microscopy (Figure 3 and Figure 4). Borrelia cells in the exponential phase are typically slender and kinked, and motile to some extent. As the cultures go into the stationary phase, round bodies are increasingly evident (Figure 5). Clinical or environmental isolates frequently contain multiple Borrelia strains and/or species. To isolate pure clones, liquid cultures can be plated to isolate monoclonal colonies (Figure 6); sometimes, multiple rounds of colony isolation may be needed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64431/64431fig01.jpg" /> 
Figure 1: BSK and MKP medium. (A) BSK (with antibiotics) with a pure culture of Borrelia burgdorferi. With or without B. burgdorferi medium remains without visible turbidity and is orange-amber (color varies slightly with ingredients). (B) MKP medium, uninoculated, thawing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64431/64431fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64431/64431fig02.jpg" /> 
Figure 2: Contaminated BSK and MKP medium with overgrowth of competing bacteria. Medium is discolored, turbid, and over time competing bacteria die and precipitate to the bottom of the tube. (A) Overgrown BSK tube. (B) Old BSK tube with precipitated contaminating bacteria. (C) MKP tube with (left) and without (middle) culture and contaminated culture (right). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64431/64431fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64431/64431fig03.jpg" /> 
Figure 3: Use of a hemocytometer to monitor Borrelia growth. Borrelia burgdorferi strain B31 (a subset indicated by arrows) viewed on a hemocytometer (one line of the small grid is indicated by the arrowhead) under 200X phase contrast. This magnification and process allow monitoring of cultures and estimation of cell density. Scale bar is 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64431/64431fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64431/64431fig04.jpg" /> 
Figure 4: Darkfield image of Borrelia burgdorferi. Darkfield image of Borrelia burgdorferi strain SCW-53, at high density in exponential phase after 5-7 days of cultivation in MKP medium (400X magnification). Scale bar is 20 &#181;m. This strain was isolated from the hard tick Ixodes affinis, collected in South Carolina, USA. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64431/64431fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64431/64431fig05.jpg" /> 
Figure 5: Borrelia in old cultures. As Borrelia cells go into the stationary phase in old cultures, round body forms are increasingly seen to replace the spirochetal form. These may be dormant forms or dead cells. Left panel, Dieterle stain; middle panel, immunohistochemistry stain; and right panel, fluorescent in situ hybridization of a Borrelia culture (presumed to be B. burgdorferi based on origin but the species and strain was not molecularly identified) inoculated with canine urine. Collection of the data in Figure 5 was approved by the Mrt. Allison University Animal Care Committee, approval number 16-1.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64431/64431fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64431/64431fig06.jpg" /> 
Figure 6: Borrelia burgdorferi sensu lato complex spirochetes. Colonies of Borrelia burgdorferi sensu lato complex spirochetes grown on solid medium in order to isolate pure clonal populations. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64431/64431fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia at JoVE.com

Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia

In vitro culture is a direct detection method for the presence of living bacteria. This protocol describes methods for the culture of diverse Borrelia spirochetes,&#160;including those of the Borrelia burgdorferi sensu lato complex, relapsing fever Borrelia species, and Borrelia miyamotoi. These species are fastidious and slow growing but can be cultured.

Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia

The Borrelia consists of three groups of species, those of the Lyme borreliosis (LB) group, also known as B. burgdorferi sensu lato (s.l.) and recently ...

cultivation-methods-spirochetes-from-borrelia-burgdorferi-sensu-lato

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

4.8K Views.  Mt. Allison University. In vitro culture is a direct detection method for the presence of living bacteria. This protocol describes methods for the culture of diverse Borrelia spirochetes, including those of the Borrelia burgdorferi sensu lato complex, relapsing fever Borrelia species, and Borrelia miyamotoi. These species are fastidious and slow growing but can be cultured.

Video: Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1. A tick-borne murine model 2 has been developed to study Lyme disease in the laboratory. While na&iacute;ve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease 3,4. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks 5. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA 6. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

We describe an in vitro method for culturing schistosomula of the flatworm parasite Schistosoma mansoni, via the harvesting and transformation of infective cercariae from the fresh water snail intermediate host Biomphalaria glabrata.

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in human non-hepatic and murine cell lines, the efficiency was very low and required long-term selection procedures using HCV replicon constructs expressing dominant antibiotic-selectable markers1-5. HCV in vitro research is therefore limited to human hepatoma cell lines permissive for virus entry and completion of the viral life cycle. Due to HCVs narrow species tropism, there is no immunocompetent small animal model available that sustains the complete HCV replication cycle 6-8. Inefficient replication of HCV in non-human cells e.g. of mouse origin is likely due to lack of genetic incompatibility of essential host dependency factors and/or expression of restriction factors.
We investigated whether HCV propagation is suppressed by dominant restriction factors in either human cell lines derived from non-hepatic tissues or in mouse liver cell lines. To this end, we developed two independent conditional trans-complementation methods relying on somatic cell fusion. In both cases, completion of the viral replication cycle is only possible in the heterokaryons. Consequently, successful trans-complementation, which is determined by measuring de novo production of infectious viral progeny, indicates absence of dominant restrictions.
Specifically, subgenomic HCV replicons carrying a luciferase transgene were transfected into highly permissive human hepatoma cells (Huh-7.5 cells). Subsequently, these cells were co-cultured and fused to various human and murine cells expressing HCV structural proteins core, envelope 1 and 2 (E1, E2) and accessory proteins p7 and NS2. Provided that cell fusion was initiated by treatment with polyethylene-glycol (PEG), the culture released infectious viral particles which infected na&iuml;ve cells in a receptor-dependent fashion.
To assess the influence of dominant restrictions on the complete viral life cycle including cell entry, RNA translation, replication and virus assembly, we took advantage of a human liver cell line (Huh-7 Lunet N cells 9) which lacks endogenous expression of CD81, an essential entry factor of HCV. In the absence of ectopically expressed CD81, these cells are essentially refractory to HCV infection 10 . Importantly, when co-cultured and fused with cells that express human CD81 but lack at least another crucial cell entry factor (i.e. SR-BI, CLDN1, OCLN), only the resulting heterokaryons display the complete set of HCV entry factors requisite for infection. Therefore, to analyze if dominant restriction factors suppress completion of the HCV replication cycle, we fused Lunet N cells with various cells from human and mouse origin which fulfill the above mentioned criteria. When co-cultured cells were transfected with a highly fusogenic viral envelope protein mutant of the prototype foamy virus (PFV11) and subsequently challenged with infectious HCV particles (HCVcc), de novo production of infectious virus was observed. This indicates that HCV successfully completed its replication cycle in heterokaryons thus ruling out expression of dominant restriction factors in these cell lines. These novel conditional trans-complementation methods will be useful to screen a large panel of cell lines and primary cells for expression of HCV-specific dominant restriction factors.

We describe two methods for conditional trans-complementation of hepatitis C virus (HCV) assembly and the completion of the full viral life cycle, which rely on heterokaryon formation. These techniques are suitable to screen for cell lines that express dominant restriction factors, which preclude production of infectious HCV progeny.

Hepatitis C virus (HCV) is a hepatotropic virus with a host-range restricted to humans and chimpanzees. Although HCV RNA replication has been observed in ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays

Borreliella burgdorferi is a bacterial pathogen with limited metabolic and genomic repertoires. B. burgdorferi transits extracellularly between vertebrates and ticks and dramatically remodels its transcriptional profile to survive in disparate environments during infection. A focus of B. burgdorferi studies is to clearly understand how the bacteria responds to its environment through transcriptional changes. In vitro transcription assays allow for the basic mechanisms of transcriptional regulation to be biochemically dissected. Here, we present a detailed protocol describing B. burgdorferi RNA polymerase purification and storage, sigma factor purification, DNA template generation, and in vitro transcription assays. The protocol describes the use of RNA polymerase purified from B. burgdorferi 5A4 RpoC-His (5A4-RpoC). 5A4-RpoC is a previously published strain harboring a 10XHis-tag on the rpoC gene encoding the largest subunit of the RNA polymerase. In vitro transcription assays consist of the RNA polymerase purified from strain 5A4-RpoC, a recombinant version of the housekeeping sigma factor RpoD, and a PCR-generated double-stranded DNA template. While the protein purification techniques and approaches to assembling in vitro transcription assays are conceptually well understood and relatively common, handling considerations for RNA polymerases often differ from organism to organism. The protocol presented here is designed for enzymatic studies on the B. burgdorferi RNA polymerase. The method can be adapted to test the role of transcription factors, promoters, and post-translational modifications on the activity of the RNA polymerase.

Essential Components of Borreliella Borrelia burgdorferi In Vitro Transcription Assays

In vitro transcription assays can decipher the mechanisms of transcriptional regulation in Borreliella burgdorferi. This protocol describes the steps to purify B. burgdorferi RNA polymerase and perform in vitro transcription reactions. Experimental approaches using in vitro transcription assays require reliable purification and storage of active RNA polymerase.

Borreliella burgdorferi is a bacterial pathogen with limited metabolic and genomic repertoires. B. burgdorferi transits extracellularly between ...

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This process has notably lower efficiencies in the Lyme disease spirochete relative to other, better-characterized Gram-negative bacteria. The rate of success of transformation is highly dependent upon having concentrated amounts of high-quality DNA from specific backgrounds and is subject to significant strain-to-strain variability. Alternative means for introducing foreign DNA (i.e., shuttle vectors, fluorescent reporters, and antibiotic-resistance markers) into B. burgdorferi could be an important addition to the armamentarium of useful tools for the genetic manipulation of the Lyme disease spirochete. Bacteriophage have been well-recognized as natural mechanisms for the movement of DNA among bacteria in a process called transduction. In this study, a method has been developed for using the ubiquitous borrelial phage &#966;BB-1 to transduce DNA between B. burgdorferi cells of both the same and different genetic backgrounds. The transduced DNA includes both borrelial DNA and heterologous DNA in the form of small shuttle vectors. This demonstration suggests a potential use of phage-mediated transduction as a complement to electroporation for the genetic manipulation of the Lyme disease spirochete. This report describes methods for the induction and purification of phage &#966;BB-1 from B. burgdorferi, the use of this phage in transduction assays, and the selection and screening of potential transductants.

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

The ability of bacteriophage to move DNA between bacterial cells makes them effective tools for the genetic manipulation of their bacterial hosts. Presented here is a methodology for inducing, recovering, and using &#966;BB-1, a bacteriophage of Borrelia burgdorferi, to transduce heterologous DNA between different strains of the Lyme disease spirochete.

Introducing foreign DNA into the spirochete Borrelia burgdorferi has been almost exclusively accomplished by transformation using electroporation. This ...

Multiplex Immunoassay for Borrelia-Specific IgG and IgM in Serum

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different antibody isotypes because they appear at different stages of the host immune response. With Lyme borreliosis, the pathogenic agent can be one of the multiple members of the Borrelia species. Therefore, correct sample classification requires evaluating the immunoreactivity against different antigens of different Borrelia species. Additionally, anti-pathogen IgG and IgM responses can have different elicitation time courses during disease progression. Here we demonstrate the development of a two-reporter multiplex immunoassay that has utility in identifying Borrelia-specific immune response in human serum samples by simultaneously evaluating both IgG and IgM immunoreactivity against different bacterial antigens in the same reaction well. This dual-reporter approach retains the analytical performance of single-reporter methods while conserving time and resources and reducing sample size requirements. This assay allows essentially double the serological information to be generated from a blood sample in half the time.

Author Spotlight: Expanding the Scope of Multiplex Immunoassays for Lyme Borreliosis Diagnostics and Pathogen Research 

A three-channel dual-reporter fluorescence flow analysis system was used to develop a bead-based multiplex immunoassay that simultaneously evaluates serum samples for IgG and IgM elicited against multiple antigens of different Borrelia species that cause Lyme borreliosis in Europe and North America.

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different ...

Human Exposure to Larval Ticks for Tickborne Disease Research

A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks

Tickborne diseases are a significant public health problem in the United States and worldwide. Ticks are obligate blood-feeding arthropods; an ixodid tick must remain attached to the skin of the host and complete its multi-day feeding process to acquire its blood meal. Exposing animals to ticks is a common practice for studying host responses to tick bites and tickborne diseases. We developed the procedure, conducted the first human research study, and published the findings on exposing human volunteers to uninfected larval Ixodes scapularis ticks. This article describes the methodology used to construct the containment dressing, how to apply and secure the ticks to the host, how to maintain the dressing, and how to remove the ticks from the host. Exposing volunteers to tick bites is an experimental procedure and must be performed under a clinical research protocol approved by the appropriate regulatory authorities. This method allows for translational research to better understand the human response to tick bites and foster the development of diagnostics, prevention, and therapies for tickborne diseases.

Author Spotlight: Controlled Human Exposure Model for Tick Research and Lyme Disease Studies

This article presents the methodology for exposing humans to larval Ixodes scapularis for clinical research. The technique is relatively simple, tolerable by the research volunteers, and can be modified according to experimental needs. Such research involving human subjects must be conducted under clinical study protocols approved by the appropriate regulatory authorities.

Tickborne diseases are a significant public health problem in the United States and worldwide. Ticks are obligate blood-feeding arthropods; an ixodid tick ...

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to humans through the bite of infected Ixodes ticks. The primary etiological agent in North America is Borrelia burgdorferi sensu stricto. As geographic risk regions expand, it is prudent to support robust surveillance programs that can measure tick infection rates, and communicate findings to clinicians, veterinarians, and the general public. The molecular technique of nested polymerase chain reaction (nPCR) has long been used for this purpose, and it remains a central, inexpensive, and robust approach in the detection of Borrelia in both ticks and wildlife. 
This article demonstrates the application of nPCR to tick DNA extracts to identify infected specimens. Two independent B. burgdorferi targets, genes encoding Flagellin B (FlaB) and Outer surface protein A (OspA), have been used extensively with this technique. The protocol involves tick collection, DNA extraction, and then an initial round of PCR to detect each of the two Borrelia-specific loci. Subsequent polymerase chain reaction (PCR) uses the product of the first reaction as a new template to generate smaller, internal amplification fragments. The nested approach improves upon both the specificity and sensitivity of conventional PCR. A tick is considered positive for the pathogen when inner amplicons from both Borrelia genes can be detected by agarose gel electrophoresis.

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR

Nested PCR is a sensitive, specific, and straightforward technique that can be applied to tick DNA extracts to probe for Borrelia burgdorferi, the causative agent of Lyme disease. The initial PCR experiment uses gene-specific primers to generate long amplicons, which then become templates for a subsequent reaction using internal primers.

Lyme disease is a serious vector-borne infection that is caused by the Borrelia burgdorferi sensu lato family of spirochetes, which are transmitted to ...

Cultivation Methods of Spirochetes from Borrelia burgdorferi Sensu Lato Complex and Relapsing Fever Borrelia

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Chapters in this video

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Two Methods of Heterokaryon Formation to Discover HCV Restriction Factors

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays

Phage-Mediated Genetic Manipulation of the Lyme Disease Spirochete Borrelia burgdorferi

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test

A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks

Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR