Name: Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient (Video) | JoVE Journal
Uploaded: 2009-11-03T16:00:00.000+00:00
Duration: 11 min 17 s
Description: Learn to purify Toxoplasma gondii oocysts using a discontinuous cesium chloride gradient method. This protocol enables high-purity isolation from cat feces for molecular biology and tissue culture experiments.

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We would like to thank Nichole Brinkman and Michael Ware for technical review of the manuscript and Dr. Abu Sayed and Linda Ransick for technical and administrative support. The United States Environmental Protection Agency through its Office of Research and Development partially funded and collaborated in the research described here under contract number EP-D-06-096 to Dynamac, Inc. and an interagency agreement number DW-12-92289801-0 to USDA. It has been subjected to Agency review and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. MJS current address: Dynamac, Inc. Cincinnati, OH.

1. General safety precautions when working with T. gondii oocysts
 <ol>
 <li>It is important to follow all safety precautions when working with T. gondii oocysts. In most healthy individuals, T. gondii infection is readily controlled by the immune system; however, a life-long infection results. Immunocompromised individuals are particularly susceptible to toxoplasmosis and should not handle T. gondii oocysts. Pregnant women should also not handle T. gondii oocysts, because infection can cause severe birth defects8. </li>
 <li>T. gondii oocysts should only be handled in a designated area and with trained personnel. Signs indicating T. gondii oocyst work is in progress must be posted to alert others entering the designated area. </li>
 <li>Wear appropriate personal protective equipment (PPE) such as a lab coat, disposable gown, disposable gloves, and proper eye protection or a face shield when handling T. gondii oocysts.</li>
 <li>Frequent glove changes are recommended. Do not handle any lab equipment with T. gondii oocyst contaminated gloves. </li>
 <li>Always use metal autoclavable trays lined with a disposable absorbent liners when working with T. gondii oocysts. Ensure all racks, tubes, etc. used are either disposable or autoclavable. </li>
 <li>All T. gondii waste must be autoclaved twice for at least one hour.</li>
 <li>All non-disposable equipment (racks, trays, etc.) must also be autoclaved twice for at least one hour.</li>
 <li>Vacuum lines used to aspirate liquids should be connected to a Vacushield&trade; filter to prevent contamination of the vacuum pump.</li>
 <li>All affected laboratory bench-tops must be disinfected after completing work with T. gondii oocysts. Freshly made 10% hypochlorite should be liberally applied to the work area and allowed dry. The area must then be rinsed well with water.</li>
 </ol>
2. Preparation of buffers and solutions
 <ol>
 <li>Prepare a 1 L 2.2 M solution of sucrose by dissolving 752.66 g of sucrose in 600 ml ddH2O. Stir and heat gently using a heated stir plate to dissolve the sucrose. Once completely dissolved, bring volume to 1 L with ddH2O. This should be followed by sterilization by autoclaving the solution for at least 20 minutes. </li>
 <li>Prepare a 1L TE buffer (50 mM Tris-HCl, 10 mM EDTA), pH 7.2 by adding 6.05 g of Tris-HCl and 3.7 g of EDTA in 700 ml of ddH2O, adjust pH to 7.2 then bring volume to 1 L with ddH2O.</li>
 <li>For the CsCl gradient, prepare a stock solution of CsCl with a specific gravity of 1.15 (1.15-CsCl) by adding 21.75 g of CsCl with 103.25 ml of TE Buffer. For Solution A, mix 30 ml of TE with 20 ml of 1.15-CsCl. For Solution B, mix 20 ml of TE with 30 ml of 1.15-CsCl and 12.5 &mu;l of phenol red solution. For Solution C, mix 10 ml of TE with 40 ml of 1.15-CsCl (Table 1).</li>
 <li>Prepare a 1L 1 N solution of sodium hydroxide (NaOH) by dissolving 40 g of NaOH, in 800 ml of ddH2O. Once dissolved, bring volume to 1 L with ddH2O.</li>
 <li> Prepare a 1 L 2% (by volume) solution of H2SO4 mixing 20 ml of H2SO4 with 980 ml of ddH2O. </li>
 </ol>
 3. Sucrose float
 <ol>
 <li>Add 10 ml of a fecal suspension of T. gondii oocysts, in 2% H2SO4, into a 50 ml conical centrifuge tube. It must be noted that fecal suspension refers to samples that have been pre-processed through a sucrose flotation procedure as previously described8. When the fecal samples are initially harvested from the infected cats they are processed through a sucrose float as described in reference 8. This additional sucrose float is necessary to further minimize fecal debris carried over to the CsCl purification process and obtain the purest T. gondii oocysts possible.</li>
 <li>Neutralize the 2% H2SO4 by adding 6 ml (3/5 volumes) of 1 N NaOH to the fecal suspension. Mix well by vortexing.</li>
 <li>Add an equal volume (16 ml) of 2.2 M sucrose creating a final concentration of 1.1 M to the fecal suspension and mix well by vortexing.</li>
 <li>Carefully overlay the sucrose/fecal suspension with 10 ml ddH2O using a 10 ml pipette. Centrifuge the suspension at 1,200 x g for 20 min at room temperature with no brake.</li>
 <li>Carefully collect the top water and interphase layers and transfer to a new 50 ml conical centrifuge tube by pipetting from the air-water interface without swirling the pipette. It is important to minimize sucrose carryover while collecting the interphase layer.</li>
 <li>Mix the remaining sucrose/fecal pellet solution by vortexing the tube.</li>
 <li>Repeat steps 3.4 and 3.5.</li>
 <li>Bring the volume of the two oocyst interphase solutions to 50 ml with ddH2O and centrifuge the tubes at 2,000 x g for 10 minutes at room temperature. </li>
 <li>Aspirate the supernatant from each tube and resuspend pellets with 5 ml TE buffer and pool the oocyst suspension together. The total pooled volume should be 10 ml. </li>
 </ol>
4. CsCl gradient
<ol>
 <li>Prepare a discontinuous CsCl gradient in a 50 ml polycarbonate Oak Ridge tube by carefully underlaying each layer using a 50 ml syringe with an 18 gauge blunt-ended, autoclavable, steel needle, and a 2 way stop cock. Note: Phenol red is added to Solution B to easily distinguish between the gradient layers (Table 1).</li>
 <li>Slowly add the following solutions to the tube in the order listed below. It should be noted that the flow rate should not exceed more than 0.5 ml/sec.
<ol>
 <li>10 ml of the TE/oocyst suspension sample</li>
 <li>8 ml Solution A</li>
 <li>8 ml Solution B</li>
 <li>8 ml Solution C</li>
</ol>
</li>
 <li>Centrifuge the Oak Ridge tube at 12,000 x g for 60 min at 4 &deg;C with no brake. Use of a fixed angle rotor is acceptable, but a high speed swinging bucket results in better separation of the oocysts from the suspension sample with minimal fecal debris smears along the side or the tube. The extent of the smears depends on the composition of the fecal suspension and thus it may be necessary to perform two CsCl gradients if the fecal suspension is extremely dirty. </li>
 <li>Following centrifugation, collect the opaque/white oocyst containing band between solutions A and B. To minimize contamination with fecal debris, go directly to the oocyst band without disturbing the gradient and collect the oocyst interphase using a 10 ml pipette. Transfer the oocyst interphase to a new 50 ml conical centrifuge tube. Try to minimize the amount of CsCl solution aspirated while collecting the oocyst interphase as it may pose a problem in pelleting the oocysts during the wash step.</li>
 <li>Wash oocysts with 30-40 ml of ddH2O. Centrifuge the tube at 2,000 x g at room temperature for 10 min with no brake. Carefully aspirate the supernatant without disturbing the oocyst pellet. Repeat the wash 2 additional times. </li>
 <li>At the end of the final wash, carefully aspirate the supernatant and resuspend the pellet in 10 ml 2% sulfuric acid and store at 4 &deg;C until use. Oocysts are now ready for further manipulation. Oocyst purity can be checked microscopically by the absence of fecal debris in the sample.</li>
</ol>

<ol><li>Dubey, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevalence+of+viable+Toxoplasma+gondii+in+beef%2C+chicken%2C+and+pork+from+retail+meat+stores+in+the+United+States%3A+risk+assessment+to+consumers%5BTitle%5D)+AND+%22J.+Parasitol%22%5BJournal%5D)">Prevalence of viable Toxoplasma gondii in beef, chicken, and pork from retail meat stores in the United States: risk assessment to consumers.</a> J. Parasitol. 91, 1082-1093 (2005).</li>
<li>Tenter, A. M., Heckeroth, A. R., Weiss, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Toxoplasma+gondii%3A+from+animals+to+humans%5BTitle%5D)+AND+%22Int.+J.+Parasitol%22%5BJournal%5D)">Toxoplasma gondii: from animals to humans.</a> Int. J. Parasitol. 30, 1217-1258 (2000).</li>
<li>Bowie, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Outbreak+of+toxoplasmosis+associated+with+municipal+drinking+water%5BTitle%5D)+AND+%22Lancet%22%5BJournal%5D)">Outbreak of toxoplasmosis associated with municipal drinking water.</a> Lancet. 350, 173-177 (1997).</li>
<li>Moura, L. de <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Waterborne+toxoplasmosis%2C+Brazil%2C+from+field+to+gene%5BTitle%5D)+AND+%22Emerg.+Infect.+Dis%22%5BJournal%5D)">Waterborne toxoplasmosis, Brazil, from field to gene.</a> Emerg. Infect. Dis. 12, 326-329 (2006).</li>
<li>Jones, J. L., Dubey, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Waterborne+toxoplasmosis+-+Recent+developments%5BTitle%5D)+AND+%22Exp.+Parasitol%22%5BJournal%5D)">Waterborne toxoplasmosis - Recent developments.</a> Exp. Parasitol. doi: 10.1016/j.exppara.2009.03.013, (2009).</li>
<li>Dumetre, A., Darde, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+to+detect+Toxoplasma+gondii+oocysts+in+environmental+samples%5BTitle%5D)+AND+%22FEMS+Microbiol.+Rev%22%5BJournal%5D)">How to detect Toxoplasma gondii oocysts in environmental samples.</a> FEMS Microbiol. Rev. 27, 651-661 (2003).</li>
<li>Dumetre, A., Darde, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+of+Toxoplasma+gondii+oocysts+by+cesium+chloride+gradient%5BTitle%5D)+AND+%22J.+Microbiol.+Methods%22%5BJournal%5D)">Purification of Toxoplasma gondii oocysts by cesium chloride gradient.</a> J. Microbiol. Methods. 56, 427-4230 (2004).</li>
<li>Dubey, J. P. Toxoplasmosis of Animals and Man. , 2nd Edition, CRC Press, Inc. Boca Raton, FL. (2009).</li>
</ol>

obtaining highly purified toxoplasma gondii oocysts by a discontinuous cesium chloride gradient

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

Toxoplasma Goia Osis are typically obtained from infected cat feces and isolated using a flotation procedure resulting in a partially purified suspension, molecular and tissue culture. Experiments on this parasite, however, require highly purified osis to achieve this. Osis are further purified using a sucrose flotation step where osis are resuspended and sucrose overlaid with water and centrifuge to separate the osis away from most of the fecal matter.The osis layer is then transferred into a new tube underlaid with cesium, chloride and centrifuged. The resulting distinct OSIS band is then collected and washed, ready to use on experiments requiring highly purified teon io assist. Hi, I'm Dr.Sarah Staggs, an employee of Dynam, a contractor for the National Exposure Research Laboratory of the US Environmental Protection Agency.Today we will show you a procedure for purifying Toxoplasma Gandhi io Oasis from the feces of laboratory infected cats using a discontinuous cesium chloride gradient. This procedure is routinely used in our laboratory to isolate OSI, assist for use of molecular biology and tissue culture experiments. So let's get started.Before beginning any work with TG O Osis, important safety considerations should be taken into account. People who are immunocompromised, pregnant, or actively trying to get pregnant should not handle the pathogen due to the risk of acquiring toxoplasmosis, which can cause illness and severe birth defects. X.To reduce the risk of infection, set aside a designated space for this procedure and post signs to warn others before they enter the room.Remember to wear a lab coat, disposable gown, disposable gloves, and proper eye protection or a face shield when handling T-One IO cysts. Be sure to change your gloves often and avoid touching any equipment with contaminated gloves. It's also important to work on metal autoclavable trays lined with disposable absorbent liners.And along those lines, take a minute to make sure all of the racks tubes and other equipment you plan to use are either disposable or autoclavable. If you're using non-disposable items, be sure to autoclave them after use for two one hour cycles. You'll also want to connect all vacuum lines to a vacuum shield filter to avoid contaminating the vacuum pump.When you're finished isolating cysts, you will need to apply a fresh 10%hypochlorite solution to your work surface. Allow it to dry and then rinse the area several times with water. Once the proper safety precautions have been put into place, you can proceed with your experiments.Prior to T Guni OYS collection, one must first prepare all of the necessary solutions for the sucrose, float and cesium chloride gradient. Centrifugation steps. The solutions that you will need include a 2.2 molar solution of sucrose sterilized by a 20 minute cycle in the autoclave, one liter of TE buffer pH 7.2 A one normal solution of sodium hydroxide, a 2%solution of sulfuric acid and a stock solution of caesium chloride with a specific gravity of one point 15.The one point 15 caesium chloride stock solution will be used to make solutions A, B, and C, which will be used to prepare the cesium chloride gradient. Solution A is made by mixing 30 milliliters of TE with 20 milliliters of one point 15 cesium chloride stock solution solution B is made by mixing 20 milliliters of TE with 30 milliliters of one point 15 cesium chloride stock solution, and then adding 12.5 microliters of pheno red solution. Finally, solution C is made by mixing 10 milliliters of TE with 40 milliliters of one point 15 cesium chloride solution.We'll begin our preparation of the sucrose float by taking a 10 milliliter fecal suspension of T osis in 2%sulfuric acid and transferring it to a 50 milliliter conical centrifuge tube. The source of the fecal matter comes from feces of infected cats that has already been processed through a sucrose flotation procedure previously described in a textbook written by Dr.JP Duby, entitled Toxoplasmosis of Animals and Humans. This additional step further reduces the amount of fecal debris in the cesium chloride purification process and helps achieve the highest purity of the T goi cysts.We then neutralize the sulfuric acid by adding three fifths volume of one normal sodium hydroxide solution to the fecal suspension. Mix the suspension well by vortexing. Next, add an equal volume of sucrose and mix the suspension well by vortexing.Using a 10 milliliter per pet, carefully overlay the sucrose fecal suspension with 10 milliliters of double distilled water. Once you are done carefully, place the tubes in the centrifuge and spin the at 1200 times G for 20 minutes at room temperature with no break after the spin. Being careful not to swell your perpe gently perpe from the air water interface to collect the top water and interface layers.It's very important to try to minimize the amount of the sucrose layer that is carried over while you're collecting the interface layer. Transfer these layers to a new 50 milliliter conical centrifuge tube. Vortex the tube to mix the remaining sucrose fecal pellet solution.Repeat the previous water overlay in centrifugation steps once more. After the spin, collect the top water and interface layers and transfer them to a new tube. Use double distilled water to bring the volume of the two oh oh cyst interface solutions to 50 milliliters.Then centrifuge the tubes at 2000 times G for 10 minutes at room temperature. Finally aspirate the S supernatant from each tube. Re suspend the pellets in five milliliters of tea buffer and pull the osis suspensions.The osis are now ready for the cesium chloride gradient step. We'll start by preparing a discontinuous cesium chloride gradient in a 50 milliliter polycarbonate Oak ridge tube to the polycarbonate tube. Add 10 milliliters of the suspension containing TE and osis using a 50 milliliter syringe with an 18 gauge blunt ended autoclavable steel needle.And a two-way stop cock slowly add the following solutions directly to the bottom of the tube. Eight milliliters of solution a eight milliliters of solution B at eight milliliters of solution C.Make sure the flow rate doesn't exceed N 0.5 milliliters per second. It's also important to note that the phenol red in solution B helps distinguish the gradient layers.Now you are ready to centrifuge the Oak ridge tube at 12, 000 times G for 60 minutes at four degrees Celsius with no break using a fixed angle, high speed rotor. Now that the centrifugation is finished, we collect the opaque white band between solutions A and B.This band contains the ooc assists. Using a 10 milliliter per pet, go directly to the ooc assists band without disturbing the gradient and collect the ooc.Try to minimize the amount of caesium chloride solution that you aspirate while you're collecting the cyst, because excess caesium chloride might make it difficult to pellet the cysts during the wash. Step later on, transfer the OSIS to a new 50 milliliter conical centrifuge tube. Now wash the O cysts with 30 to 40 milliliters of double distilled water and then centrifuge the tube at 2000 times G at room temperature for 10 minutes with no break, carefully aspirate the SUP natant being careful not to disturb the O cyst pellet.Repeat the wash two more times. Once the final washers have finished, carefully aspirate the sup natant and resuspend the pellet in 10 milliliters of 2%sulfuric acid. Check the OSIS under the microscope to ensure that all fecal debris has been successfully removed from the sample.The osis are now ready for further manipulation, or you can store them at four degrees Celsius until you're ready to use them. Here is a look at what un purified osis look like using a differential interference contrast microscope at 400 x magnification. Notice the presence of large amounts of fecal contaminants in the field making it very difficult to identify the OSI assists.Here is what the OISS look like under 400 x after the cesium chloride gradient purification protocol. Notice the absence of fecal debris. We've just shown you how to purify Toxoplasma Gandhi Iasis from the feces of laboratory infected cats using a discontinuous cesium chloride gradient.When doing this procedure, it's important to remember to follow all safety guidelines and to always be careful when underlaying the cesium chloride gradient by adding each layer very slowly and trying not to disturb or mix the gradient. So that's it. Thanks for watching and good luck with your experiments.

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Learn to purify Toxoplasma gondii oocysts using a discontinuous cesium chloride gradient method. This protocol enables high-purity isolation from cat feces for molecular biology and tissue culture experiments.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

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