This work was supported by grant R01 AI141958 from the United States National Institutes of Health/NIAID. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Scalable In Vitro Cultivation of T. pallidum Using Mammalian Cell Co-Culture

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+TprK+profiling+of+in+vivo+and+in+vitro-propagated+Treponema+pallidum+subsp.+pallidum+reveals+accumulation+of+antigenic+variants+in+absence+of+immune+pressure.">Longitudinal TprK profiling of in vivo and in vitro-propagated Treponema pallidum subsp. pallidum reveals accumulation of antigenic variants in absence of immune pressure.</a> PLoS Negl Trop Dis. 15 (9), e0009753 (2021).</li><li>De Lay, B. D., Cameron, T. A., De Lay, N. R., Norris, S. J., Edmondson, D. 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The authors have no conflicts of interest to disclose.

The Sf1Ep-TpCM-2 system is the first available procedure that promotes the continuous in vitro culture of T. pallidum. The system is complex due to the extreme growth requirements of this organism: 1) complex nutritional needs because of limited biosynthetic capabilities; 2) a poorly understood requirement for small quantities of oxygen, despite high sensitivity to reactive oxygen species9,10,16,36; and 3) the current need for the presence of Sf1Ep cells. While it is tempting to &#39;cut corners&#39; on the procedure, it is recommended that the steps be followed carefully until a successful long-term culture is achieved prior to trying modifications. As additional information about the metabolic requirements of T. pallidum accumulates, it may be possible to develop axenic conditions that do not require the presence of Sf1Ep cells. However, the in vitro growth rate will likely remain slow (with a minimum doubling time of 33 h to 46 h, depending on the strain)17,18, given that T. pallidum multiplies at an estimated doubling time of 30 h to 33 h even during mammalian infection37,38. As with any bacterial culture, it is recommended that low passage stocks be maintained and that experiments be carried out with T. pallidum cultures that are less than 10 passages from these stocks to avoid &#39;genetic drift&#39; and associated phenotypic changes due to mutations.
Sf1Ep cells apparently provide essential nutrients or enzymatic activities to the treponemes. However, they also consume nutrients (such as glucose and oxygen) and may produce toxic conditions such as low pH9,16,39. Therefore, there is a balancing act between having sufficient Sf1Ep cells to support T. pallidum multiplication and preventing mammalian cell overgrowth and toxicity. High passage numbers of Sf1Ep cells tend to be faster growing and at times, lose the ability to support T. pallidum multiplication. As such, Sf1Ep passage number should be monitored, and cell stocks should be replaced with low-passage frozen preparations periodically. The presence of Sf1Ep cells also complicates the study of T. pallidum properties such as DNA, RNA, and protein content, and enzyme activities. Removal of the rabbit cells is possible to some extent using repeated low-speed centrifugation (100 x g for 5 min) or more effectively using Percoll or Hypaque gradients40,41. However, the gradient centrifugation methods are generally only effective with high numbers of T. pallidum. Alternative methods for propagating T. pallidum are limited to infection of laboratory animals such as rabbits13,14. This approach has ethical considerations and has become increasingly expensive; however, the rabbit model is very useful for studying T. pallidum pathogenesis and host immune responses. In addition, there are likely some differences in gene expression, growth or behavior of T. pallidum during in vitro culture and rabbit infection27.
At the time of this report, the Sf1Ep-TpCM-2 system has been established in at least 6 research groups in the United States and Europe and has resulted in 16 publications with topics ranging from T. pallidum basic biology and genetics to antimicrobial susceptibility. The value of in vitro culture in studying this enigmatic pathogen will likely increase with expanded usage and future improvements.

Treponema pallidum is a species of spiral-shaped bacteria (called spirochetes) that causes syphilis and related infections in humans and other primates. Syphilis is a serious disease with long-term effects on infected individuals, and it is estimated that over 8 million new cases of syphilis occur worldwide each year1. T. pallidum has been subdivided into three subspecies based on the diseases they cause in humans as well as minor genetic differences: subspecies pallidum (which causes the sexually transmitted disease syphilis), subspecies pertenue (yaws), and subspecies endemicum (causing bejel or endemic syphilis)2,3. T. pallidum subsp. pertenue also causes infections in baboons, chimpanzees, and other primates. A closely related organism called Treponema paraluiscuniculi (also called Treponema paraluisleporidarum) causes an infection in rabbits and hares4,5. All of these bacteria are very closely related, with greater than 98% DNA sequence identity at the genome level6,7,8. They each have a single, small circular chromosome about 1.14 million base pairs (Mb) in size. The members of this T. pallidum group are found only in association with their mammalian hosts; as such, they are obligate pathogens that are dependent on their host species for survival and growth9,10.
Attempts to culture T. pallidum in vitro began shortly after its identification by Schaudinn and Hoffman in 190511,12. However, these efforts failed to lead to consistent, reproducible growth of the organism. As a result, T. pallidum research studies required propagation of the organism through the experimental infection of laboratory animals, most commonly the rabbit13,14. In 1981, Fieldsteel et al.15 introduced a tissue culture system that promoted the multiplication of T. pallidum strains for a period of up to 2 weeks. This system involved co-culture of T. pallidum with Sf1Ep cottontail rabbit epithelial cells in a modified tissue culture medium (T. pallidum Culture Medium 1, TpCM-1) based on Eagle&#39;s Minimum Essential Medium (MEM) and 20% fetal bovine serum (FBS). Other culture conditions required were incubation at 34 &#176;C in an atmosphere containing 1.5% O2 and 5% CO29,16. In this system, T. pallidum attaches to the Sf1Ep cells and multiplies when in close association with the mammalian cell surface. Despite many subculture attempts and other modifications, the Fieldsteel et al. system failed to promote continuous in vitro growth.
In 2018, our laboratory reported that the use of a modified medium called TpCM-2 (in which Eagle&#39;s MEM was replaced with a more complex tissue culture medium, CMRL 1066) provided T. pallidum the required nutrients to permit consistent long-term culture17. To date, this modification has led to a consistent, continuous culture of at least 5 strains of T. pallidum subsp. pallidum (Nichols, SS14, Mexico A, UW231B, and UW249B) and one strain of T. pallidum subsp. endemicum (Bosnia A)18,19. As an example, the Nichols strain has now been cultured continuously in vitro for over 6 years. Thus far, attempts to culture yaws isolates (T. pallidum subsp. pertenue) or T. paraluiscuniculiin vitro have been unsuccessful18. The TpCM-2 system still requires the presence of Sf1Ep cells, low oxygen concentrations, and incubation at 34 &#176;C, making the system more complex than most bacterial culture techniques. However, this modified T. pallidum culture system has been useful for further defining the bacterium&#39;s growth requirements18, determining minimal inhibitory concentrations (MICs) of antimicrobial compounds and peptides20,21,22,23,24,25, propagating new strains from patient tissue aspirates26, isolating clonal populations of the organism27, characterizing the tprK antigenic variation system27,28, examining gene expression29, and performing mutational analysis30,31,32.
Here, we describe the current methods for cultivating T. pallidum in vitro. We hope this information will help facilitate the more widespread application of this in vitro culture technique to the improved diagnosis, treatment, and prevention of syphilis and related treponemal infections.

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	<tbody>
		<tr>
			<td>0.5 M EDTA, pH 8.0</td>
			<td>Sigma&#160;</td>
			<td>E8008</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Earle&#8217;s Balanced Salts, w/o Mg2+, Ca2+</td>
			<td>Gibco</td>
			<td>14155063</td>
			<td></td>
		</tr>
		<tr>
			<td>15 and 50 mL conical sterile disposable centrifuge tubes&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL cryogenic vials&#160;</td>
			<td>Corning</td>
			<td>430659</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plates for T. pallidum cultivation&#160;</td>
			<td>Falcon</td>
			<td>353046</td>
			<td>The plates must have low evaporation lids.</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm2 tissue culture flasks with vented caps</td>
			<td>Corning</td>
			<td>43061U</td>
			<td></td>
		</tr>
		<tr>
			<td>93.5% nitrogen, 5% CO2, and 1.5% oxygen for pre-equilibrating medium and cultures</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>95% nitrogen and 5% CO2 for pre-equilibrating medium and cultures</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well low evaporation clear, flat-bottom tissue culture-treated microplates</td>
			<td>Corning Falcon</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable multi-channel pipette with 200 ul capacity&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Optional, but very helpful for cloning</td>
		</tr>
		<tr>
			<td>Cell culture grade water</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td></td>
		</tr>
		<tr>
			<td>CMRL 1066 without L-Glutamine or Phenol Red</td>
			<td>US Biological</td>
			<td>C5900-03A</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 for tri-gas and tissue culture incubators&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic liquid nitrogen cell culture storage tank</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G6152</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable filter units, 0.2 &#181;m , &#62; 100 mL capacity</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable pipets: 25 mL, 10 mL, 5 mL, aspirating</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Mannitol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M1902</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO (sterile cell culture grade )</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle&#8217;s MEM</td>
			<td>Sigma-Aldrich</td>
			<td>M4655</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum, heat inactivated&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F4135</td>
			<td>We highly recommend this product. Must pre-screen for T. pallidum culture compatibility if using a different brand or catalog number.</td>
		</tr>
		<tr>
			<td>Freezer with capability of maintaining -70 &#176;C or -80 &#176;C</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For storage of T. pallidum; liquid nitrogen storage may be used instead</td>
		</tr>
		<tr>
			<td>Freezing medium (Sf1Ep medium + 10% [v/v] DMSO)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas cylinders with appropriate fittings</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>GasPak 150 vented anaerobic jar (Brewer Jar)</td>
			<td>Fisher Scientific</td>
			<td>11-816</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Helber counting chambers with Thoma rulings</td>
			<td>Hawksley Medical and Laboratory Equipment</td>
			<td></td>
			<td>For quantitating T. pallidum</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>&#160;For Sf1Ep cell quantitation</td>
		</tr>
		<tr>
			<td>Incubator tank switch</td>
			<td>NuAire&#160;</td>
			<td>NU-1550 TankGuard Automatic CO2 Incubator Tank Switch</td>
			<td>Optional, but very helpful in maintaining appropriate O2 conditions.</td>
		</tr>
		<tr>
			<td>Inverted microscope with phase contrast optics</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>For viewing Sf1Ep cell cultures</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Histidine&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>H6034</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope with darkfield condensor</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>The microscope should have a 40x objective and 15x eyepieces.</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-Aldrich</td>
			<td>M3183</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel adapter for aspirator&#160;</td>
			<td>Integra</td>
			<td>155520</td>
			<td>Optional, but useful for cloning</td>
		</tr>
		<tr>
			<td>NaHCO3 (7.5%)</td>
			<td>Sigma-Aldrich</td>
			<td>S8761</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen for tri-gas incubator</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Resazurin</td>
			<td>Sigma-Aldrich</td>
			<td>R7017</td>
			<td></td>
		</tr>
		<tr>
			<td>Sf1Ep (NBL-11) cells</td>
			<td>American Type Culture Collection</td>
			<td>CCL-68</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile PBS (without calcium chloride and magnesium chloride)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile reagent reservoirs, 50 or 100 mL size</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>T. pallidum sample, frozen or fresh&#160;</td>
			<td></td>
			<td></td>
			<td>from a rabbit infection or in vitro culture</td>
		</tr>
		<tr>
			<td>Tissue culture incubator maintained at 37 &#176;C, 5% CO2</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-gas tissue culture incubator maintained at 34 &#176;C, 5% CO2, 1.5% O2</td>
			<td>Thermofisher</td>
			<td>Heracell&#8482; VIOS 160i Tri-Gas CO2 Incubator</td>
			<td>Optional; anaerobic jars may be used instead (see Ref. 17)</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum source (e.g. house vacuum), vacuum tubing, vacuum gauge, and connectors</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, suitable for cell culture, filter-sterilized, purified</td>
			<td>Sigma-Aldrich</td>
			<td>W3500</td>
			<td>Recommended for medium preparation; decreases culture variability</td>
		</tr>
	</tbody>
</table>

procedures for in vitro cultivation of treponema pallidum, the syphilis spirochete

For over a century, Treponema pallidum subsp. pallidum, the spiral-shaped bacterium that causes syphilis, could only be propagated by inoculation and harvest of the organisms from rabbit testes. In 2018, we described a method to continuously cultivate T. pallidumin vitro. This system utilizes co-culture with rabbit epithelial cells (Sf1Ep cells) in a serum-containing tissue culture medium called TpCM-2. The T. pallidum doubling time in culture is similar to that estimated to occur during natural infection (about 33-45 h). The organism can be cultured continuously with a standard passage time of 1 week in a low oxygen (1.5%) environment at 34 &#176;C. This article contains the protocols for culturing T. pallidum, methods for growing and maintaining the required tissue culture cells, and the technique for generating isogenic strains by limiting dilution. The ability to grow T. pallidum in vitro provides new experimental avenues to study and understand this enigmatic organism.

Using the described conditions, T. pallidum typically retains&#62;90% motility and multiplies logarithmically with a doubling time of 33 h to 45 h for approximately 7 days before entering the stationary phase (Figure 3). Over the course of 1 week, the spirochetes undergo approximately 4-5 doublings (Figure 4). ). In addition, different strains of T. pallidum may grow at different rates. Strains of the SS14 group of T. pallidum tend to have slower doubling times than those of the Nichols group17.
Feeding of cultures may extend culture time by several days but the Sf1Ep cell layer often fails after a week of culture. Furthermore, the treponemes reach an upper limit of organisms of about 5 x 107/mL. Cultures transferred at 7 day intervals typically continue logarithmic multiplication with little or no lag phase. Organisms in the stationary phase often become difficult to pass.
Most T. pallidum are attached to the Sf1Ep cells. However, enough T. pallidum remains in the supernatant that medium samples can be removed periodically to check for viability and multiplication. If careful quantitation is required, the volume of the removed medium should be measured, the number of T. pallidum quantitated, and the total number of organisms removed added to the final counts at harvest.
In previous studies, the cloning efficiency (number of cultures positive per organism inoculated) were 12.5% for 2 T. pallidum inoculated per well and 6.7% for 0.5 T. pallidum inoculated per well27. Thus, it is likely that any positive well represents the outgrowth of a single organism at either of these inocula. However, the clonality of the resulting populations must be verified by examining the culture for homogeneity at sites that are heterogeneous in the parent culture. The most definitive way to demonstrate that the culture is isogenic is through whole genome sequencing27.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66880/66880fig01.jpg" /> 
Figure 1: Flow chart of the T. pallidumin vitro cultivation procedure. This figure has been reprinted with permission from Edmondson and Norris (2021)19. <a href="https://www.jove.com/files/ftp_upload/66880/66880fig01large.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/66880/66880fig02.jpg" /> 
Figure 2: Diagram of the system to equilibrate T. pallidum culture reagents to a low-oxygen environment. A Brewer jar vent is connected via a T-joint to a vacuum source (such as a house vacuum) and to gas cylinders containing custom gas mixtures (5% CO2, balance nitrogen and 1.5% O2, 5% CO2, balance nitrogen). An in-line vacuum gauge measures the vacuum drawn in the jar. The vacuum is drawn in the jar to approximately -58 kPa. The evacuated jar is then slowly re-filled with the gas mixtures. The Brewer jar is refilled three times with 5% CO2, balance nitrogen before a final evacuation, and refilled with 95% N2, 5% CO2, 1.5% O2. Cultures or media are then removed from the jar and quickly transferred to the low-oxygen incubator. Alternately, the tubing between the Brewer jar and the first T-joint can be clamped tightly, the tubing disconnected from the T-joint, and the entire Brewer jar can be transferred to a 34 &#176;C incubator. This figure has been reprinted with permission from Edmondson and Norris (2021)19. <a href="https://www.jove.com/files/ftp_upload/66880/66880fig02large.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/66880/66880fig03.jpg" /> 
Figure 3: Growth curves of T. pallidum cultured with Sf1Ep cells with TpCM-2 medium. Parallel triplicate cultures were seeded with T. pallidum. Replicates were harvested at each time point; the results represent the mean + SEM for these cultures. (A) The changes in T. pallidum per culture and (B) percent motility are shown. This figure has been adapted with permission from Edmondson et al.18. <a href="https://www.jove.com/files/ftp_upload/66880/66880fig03large.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/66880/66880fig04.jpg" /> 
Figure 4: Example of passage of in vitro culture of T. pallidum, Nichols strain. Parallel triplicate cultures were seeded with T. pallidum and passed weekly using a 1:20 dilution. The sawtooth plot shows the numbers of T. pallidum per culture and the number transferred to new cultures at each time point. Results represent the mean &#177; SEM for three biological replicates. <a href="https://www.jove.com/files/ftp_upload/66880/66880fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Medium volume and seeding ratios for culture vessels. <a href="https://www.jove.com/files/ftp_upload/66880/Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Media for T. pallidum cultivation. All media should be filter sterilized after preparation. Sf1Ep medium may be stored at 4 &#176;C for up to two months. TpCM-2 is typically made one day prior to use. The dissociation medium should be aliquoted and frozen. <a href="https://www.jove.com/files/ftp_upload/66880/Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

Author Spotlight: Advancing Syphilis Research &#8212; Innovations in Treponema pallidum Cultivation and Genetic Engineering

This protocol describes in vitro cultivation of the syphilis pathogen Treponema pallidum subsp. pallidum in co-culture with mammalian cells. The method is scalable; it can be used to produce large quantities of T. pallidum and to generate clonal cultures.

Procedures for In Vitro Cultivation of Treponema pallidum, the Syphilis Spirochete

For over a century, Treponema pallidum subsp. pallidum, the spiral-shaped bacterium that causes syphilis, could only be propagated by inoculation and ...

procedures-for-vitro-cultivation-treponema-pallidum-syphilis

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JoVE Journal

Immunology and Infection

1.2K Views.  University of Texas Health Science Center at Houston - McGovern Medical School. This protocol describes in vitro cultivation of the syphilis pathogen Treponema pallidum subsp. pallidum in co-culture with mammalian cells. The method is scalable; it can be used to produce large quantities of T. pallidum and to generate clonal cultures.

Video: Author Spotlight: Advancing Syphilis Research — Innovations in Treponema pallidum Cultivation and Genetic Engineering

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

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 ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor treatment options exist. The hallmarks of these laboratories are: custom-designed airtight doors, dedicated supply and exhaust airflow systems, a negative-pressure environment, and mandatory use of positive-pressure (&#8220;space&#8221;) suits. The risk for laboratory specialists working with highly pathogenic agents is minimized through rigorous training and adherence to stringent safety protocols and standard operating procedures. Researchers perform the majority of their work in BSL-2 laboratories and switch to BSL-4 suit laboratories when work with a high-consequence pathogen is required. Collaborators and scientists considering BSL-4 projects should be aware of the challenges associated with BSL-4 research both in terms of experimental technical limitations in BSL-4 laboratory space and the increased duration of such experiments. Tasks such as entering and exiting the BSL-4 suit laboratories are considerably more complex and time-consuming compared to BSL-2 and BSL-3 laboratories. The focus of this particular article is to address basic biosafety concerns and describe the entrance and exit procedures for the BSL-4 laboratory at the NIH/NIAID Integrated Research Facility at Fort Detrick. Such procedures include checking external systems that support the BSL-4 laboratory, and inspecting and donning positive-pressure suits, entering the laboratory, moving through air pressure-resistant doors, and connecting to air-supply hoses. We will also discuss moving within and exiting the BSL-4 suit laboratories, including using the chemical shower and removing and storing positive-pressure suits.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 1. Biosafety Level 4 Suit Laboratory Suite Entry and Exit Procedures

Although researchers are generally knowledgeable about procedures and safety precautions required for biosafety level 1 or 2 (BSL-1/2) experiments, they may not be familiar with experimental procedures in BSL-4 suit laboratories. This article provides a detailed visual demonstration of BSL-4 suit laboratory systems check, laboratory entry, movement, and exit procedures.

Biosafety level 4 (BSL-4) suit laboratories are specifically designed to study high-consequence pathogens for which neither infection prophylaxes nor ...

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that employs multiple immunomodulatory mechanisms to establish chronic infection in mice and closely resembles prevalent human helminth infections. H. polygyrus has been studied extensively in the field of helminth-derived immune regulation and has been found to potently suppress experimental models of allergy and autoimmunity (both with active infection and isolated secreted products). The protocol described in this paper outlines management of the H. polygyrus life cycle for consistent production of L3 larvae, recovery of adult parasites, and collection of their excretory-secretory products (HES).

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that ...

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 4. Medical Imaging Procedures

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by high-consequence pathogens has not been possible. In 2014, the National Institutes of Health, National Institute of Allergy and Infectious Diseases, Integrated Research Facility at Fort Detrick opened an Animal Biosafety Level 4 (ABSL-4) facility to assess the clinical course and pathology of infectious diseases in experimentally infected animals. Multiple imaging modalities including computed tomography (CT), magnetic resonance imaging, positron emission tomography, and single photon emission computed tomography are available to researchers for these evaluations. The focus of this article is to describe the workflow for safely obtaining a CT image of a live guinea pig in an ABSL-4 facility. These procedures include animal handling, anesthesia, and preparing and monitoring the animal until recovery from sedation. We will also discuss preparing the imaging equipment, performing quality checks, communication methods from "hot side" (containing pathogens) to "cold side," and moving the animal from the holding room to the imaging suite.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 4. Medical Imaging Procedures

Here, we present an overview of the preparation and animal handling procedures required to safely perform medical imaging in an animal biosafety level 4 laboratory. Computed tomography of a mock-infected guinea pig illustrates these procedures that may be used to evaluate the disease caused by a high consequence pathogen.

Medical imaging using animal models for human diseases has been utilized for decades; however, until recently, medical imaging of diseases induced by ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...